Fructose-1,6-Bisphosphatase Inhibits ERK Activation and Bypasses

Published OnlineFirst July 18, 2017; DOI: 10.1158/0008-5472.CAN-16-3143 Cancer Tumor and Stem Cell Biology Research

Fructose-1,6-bisphosphatase Inhibits ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking IQGAP1–MAPK Interaction Xin Jin1,2, Yunqian Pan2, Liguo Wang3, Tao Ma3, Lizhi Zhang4, Amy H. Tang5,6, Daniel D. Billadeau2,7, Heshui Wu8, and Haojie Huang2,7

Abstract

Dysregulation of the MAPK pathway correlates with progres- decreased FBP1 expression induced pERK1/2 levels in PDAC sion of pancreatic ductal adenocarcinoma (PDAC) progression. cell lines and correlated with increased pERK1/2 levels in IQ motif containing GTPase-activating protein 1 (IQGAP1) is a patient specimens. Treatment with gemcitabine caused unde- MAPK scaffold that directly regulates the activation of RAF, MEK, sirable activation of ERK1/2 in PDAC cells, but cotreatment and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in with the FBP1-derived small peptide inhibitor FBP1 E4 over- gluconeogenesis, is transcriptionally downregulated in various came gemcitabine-induced ERK activation, thereby increasing cancers, including PDAC. Here, we demonstrate that FBP1 acts as a the anticancer efﬁcacy of gemcitabine in PDAC. These ﬁndings negative modulator of the IQGAP1–MAPK signaling axis in PDAC identify a primary mechanism of resistance of PDAC to stan- cells. FBP1 binding to the WW domain of IQGAP1 impeded dard therapy and suggest that the FBP1–IQGAP1–ERK1/2 sig- IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a naling axis can be targeted for effective treatment of PDAC. manner independent of FBP1 enzymatic activity. Conversely, Cancer Res; 77(16); 1–14. 2017 AACR.

Introduction Although many therapeutic agents, such as gemcitabine and nab- paclitaxel, have been developed for pancreatic cancer treatment, Pancreatic ductal adenocarcinoma (PDAC) is one of the lead- PDAC is generally insensitive to both chemo- and radiotherapy. ing causes of cancer-related death worldwide (1). It is estimated Therefore, there is an urgent medical need to develop novel that more than 330,000 people are diagnosed with pancreatic therapeutics for pancreatic cancer treatment. cancer annually (2). Despite its relatively low epidemiologic Activation mutations in RAS are very common, with the fre- ranking, PDAC is notorious for its ability to evade early diagnosis quency as high as 90% in PDAC (4). Dysregulation of MAPK and high capability to invade and metastasize. The prognosis of pathway correlates with progression of PDAC. Increased ERK PDAC remains poor, and the occurrence and death rate of this phosphorylation has been frequently detected in PDAC (5). The disease remain largely unchanged after decades of studies (3). scaffold protein IQ-domain GTPase-activating protein 1 (IQGAP1) contains multiple protein-interacting domains and participates in multiple cellular functions, such as cell polariza- 1Department of Digestive Surgical Oncology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. tion and directional migration, adhesion, growth, and transfor- 2Department of Biochemistry and Molecular Biology, Mayo Clinic College of mation. IQGAP1 overexpression is highly correlated with pan- Medicine, Rochester, Minnesota. 3Department of Medical Informatics and Sta- creatic cancer cell metastasis. Particularly, IQGAP1 functions as a tistics, Mayo Clinic College of Medicine, Rochester, Minnesota. 4Department of key scaffold for the MAPK pathway by directly binding to and Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, 5 modulating the activities of RAF, MEK, and ERK (6, 7). Impor- Minnesota. Department of Microbiology and Molecular Cell Biology, Eastern tantly, it has been shown previously that IQGAP1 is required Virginia Medical School, Norfolk, Virginia. 6Leroy T. Canoles Jr. Cancer Center, Eastern Virginia Medical School, Norfork, Virginia. 7Mayo Clinic Cancer Center, in RAS-driven tumorigenesis in mouse and human tissues. Mayo Clinic College of Medicine, Rochester, Minnesota. 8Department of Pan- ERK1/2 bind to the WW domain of IQGAP. A peptide derived creatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of from the WW domain disrupts the interaction of IQGAP1–ERK1/2 Science and Technology, Wuhan, Hubei, China. and inhibits pancreas tumorigenesis (8). The scaffold–kinase Note: Supplementary data for this article are available at Cancer Research interaction represents a promising therapeutic target to treat Online (http://cancerres.aacrjournals.org/). pancreatic cancer. Corresponding Authors: Haojie Huang, Mayo Clinic, 200 First Street SW, Gugg Expression of FBP1 is downregulated in various types of cancer, 1311B, Rochester, MN 55905. Phone: 507-293-1712; Fax: 507-293-3071; E-mail: including breast cancer, hepatocellular carcinoma, pancreatic [email protected]; and Heshui Wu, Union Hospital, Tongji Medical Col- cancer, renal carcinoma, lung cancer, among others (9–13). FBP1 lege, Huazhong University of Science and Technology, 1277 Jiefang Road, acts as a tumor suppressor, and downregulation of FBP1 is Wuhan, Hubei 430022, China. E-mail: [email protected] associated with tumor progression and poor prognosis in hepa- doi: 10.1158/0008-5472.CAN-16-3143 tocellular carcinoma and pancreatic carcinoma. It has been 2017 American Association for Cancer Research. reported that FBP1 suppresses tumor progression mainly by

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inhibition of the Warburg effect (10). Further studies show that it glutathione-Sepharose beads (GE Healthcare Life Sciences). After also suppresses renal carcinoma cell growth by inhibiting the washing with lysis buffer, the beads were incubated with cell function of transcription factor HIF1a (12). In the current study, lysates for 4 hours. The beads were then washed four times with we identified a novel role of FBP1 in inhibition of tumor pro- binding buffer and resuspended in sample buffer. The bound gression. We demonstrated that FBP1 inhibits the activity of proteins were subjected to SDS/PAGE and Western blotting. ERK1/2 in a manner independent of its enzymatic activity. We further showed that binding to the WW domain of IQGAP1 Western blotting enables FBP1 to inhibit the IQGAP1–ERK1/2 interaction, Cells were harvested and lysed by cell lysis buffer (50 mmol/L IQGAP1-dependent activation of ERK1/2, and growth and che- Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium moresistance of PDAC cells. deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktails) on ice for more than 15 minutes. Cell lysate was centrifuged for 10 Materials and Methods minutes at 13,000 rpm at 4C, and the supernatant was trans- Cell lines, cell culture, and transfection ferred to a new tube for BCA protein quantification assay. Equal The pancreatic cancer cell lines PANC-1 and MIA PaCa-2 were amounts of protein sample were added into 4 sample buffer and obtained from Dr. D.D. Billadeau at Mayo Clinic (Rochester, MN) boiled for 5 minutes. The sample was subjected to SDS-PAGE in 2015 and authenticated via STR profiling in 2017 (IDEXX analysis and transferred to nitrocellulose membrane. The mem- BioResearch). These cell lines were cultured in DMEM supple- brane was blocked by 5% milk for 1 hour at room temperature mented with 10% FBS. Cells were cultured at 37C supplied with and incubated with primary antibody at 4C overnight. The 5% CO2. Mycoplasma contamination was regularly examined membrane was washed six times with 1 TBST and incubated using Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich). with horseradish peroxidase–conjugated secondary antibodies Plasmocin (InvivoGen) was routinely added to the cell culture for 1 hour at room temperature. The protein was visualized by medium to prevent or eliminate mycoplasma contamination. SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Transfections were performed by using Lipofectamine 2000 Scientific). (Thermo Fisher Scientific). Approximately 75% to 95% transfection efficiencies were routinely achieved. RNAi Lentivirus-based control and gene-specific small hairpin RNAs fi fi Tandem af nity puri cation (shRNA) were purchased from Sigma-Aldrich. Lipofecta- 293T cells were transfected with SFB-tagged FBP1 or empty mine2000 was used to transfect 293T cells with shRNA plasmid vector. Twenty-four hours post transfection, cells were lysed by and viral packaging plasmids (pVSV-G and pEXQV). Twenty-four NETN buffer (20 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 1 hours posttransfection, medium was replaced with DMEM con- b mmol/L EDTA, 0.5% Nonidet P-40, 50 mmol/L -glyceropho- taining 10% FBS with 1:100 of sodium pyruvate. Forty-eight m sphate, 10 mmol/L NaF, and 1 g/mL pepstatin A) at 4 C for 3 hours posttransfection, virus culture medium was collected and hours. The supernatant was collected for incubation with strep- m added to PANC-1 or MIA PaCa-2 cells treated with 12 g/mL of tavidin sepharose beads (GE Healthcare Sciences) at 4 C over- polybrene. PANC-1 or MIA PaCa-2 cells were harvested 48 hours night. The next day, the beads were washed with NETN buffer for after virus infection and puromycin selection. shRNA sequence fi ve times and then eluted by 2 mmol/L biotin (Sigma-Aldrich) for information is provided in Supplementary Table S1. 1 hour at 4C twice. The elution products were incubated with S- protein agarose beads (Novagen) at 4C overnight, and after washing three times, the products bound to S-protein agarose Cell infection with lentiviral expression vectors beads were subjected to SDS-PAGE and analyzed by silver staining The pTsin lentiviral expression vector was used to generate and mass spectrometry. lentiviral plasmids for pTsin-SFB-FFBP1 wild type (WT), pTsin- SFB-FBP1 G260R, and pTsin-SFB-FBP1 E4. shRNA-resistant expres- Coimmunoprecipitation sion vector for pTsin-SFB-FBP1 WT or mutants were generated Cells were harvested and lysed by IP buffer (50 mmol/L Tris- using the KOD-Plus Mutagenesis Kit (Toyobo). Lipofectamine HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium 2000 was used to transfect 293T cells with pTsin expression d deoxycholate, and 1% protease inhibitor cocktails) on ice for plasmid and viral packaging plasmids (pHR' CMV 9.8 and more than 15 minutes. Cell lysate was centrifuged for 10 minutes pVSV-G). Twenty-four hours after transfection, medium in 293T at 13,000 rpm at 4C, and the supernatant was transferred to a new cells was replaced with fresh DMEM containing 10% serum plus tube. The supernatant was incubated with primary antibodies and 1:100 sodium pyruvate. Twenty-four hours after sodium pyruvate protein A/G agarose beads (Thermo Fisher Scientific) with gentle treatment, cell culture medium was collected and added to PANC-1 m rocking at 4C overnight. The next day, the pellet was washed six or MIA PaCa-2 cells, followed by treatment with 12 g/mL of times with 1 IP buffer on ice and then subjected to Western polybrene. Stable cell lines were selected using puromycin. blotting. MTS assay Glutathione S-transferase pull-down assay Cell viability was measured using the MTS assay according to Cells were lysed with cell lysis buffer [20 mmol/L Tris-HCl (pH the manufacturer's instructions (Promega). PANC-1 and MIA 7.5), 150 mmol/L NaCl, 0.1% Nonidet P40, 1 mmol/L DTT PaCa-2 cells were seeded in 96-well plates with 100 mL of culture (dithiothreitol), 10% glycerol, 1 mmol/L EDTA, 2.5 mmol/L medium. Each well was added with 20 mL of CellTiter 96R MgCl2, and 1 mg/mL leupeptin] for 30 minutes at 4C. Glutathi- AQueous One Solution Reagent (Promega), and absorbance was one S-transferase (GST) fusion proteins were immobilized on measured in a microplate reader at 490 nm.

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FBP1 Inhibits IQGAP1 Activation of ERK

Glucose consumption and lactate production measurement Results assay FBP1 binds to the scaffold protein IQGAP1 Cells were plated in 6-well plates and cultured in DMEM To search for novel functions of FBP1, we constructed a FBP1 medium without phenol red (Thermo Fisher Scientific). Twen- mammalian expression vector (SFB-FBP1), which contains S, ty-four hours after plasmid transfection or 48 hours after lenti- Flag, and Biotin-binding-protein-(streptavidin)-binding-peptide virus infection, the spent medium was collected. Glucose con- tags. This plasmid and the backbone vector were individually centration of the spent medium was measured using a Glucose transfected into 293T cells, and cell extracts were prepared for (GO) Assay Kit according to the manufacturer's instructions tandem affinity protein purification and mass spectrometry. A (Sigma-Aldrich). Glucose consumption was the difference of number of binding partners of FBP1, including IQGAP1, were glucose concentration between the spent medium and unused identified (Fig. 1A; Supplementary Table S2; ref. 16). Given that medium. Lactate levels were measured using a Lactate Assay Kit IQGAP1 is a scaffold protein that plays an important role in (Eton Bioscience). The glucose consumption and lactate genera- activation of the RAF–MEK–ERK pathway and tumorigenesis in tion were normalized to cell numbers. PDAC (8, 17), we were interested in exploring the molecular basis and biological impact of the interaction between FBP1 and Detection of apoptosis using Annexin V assay and flow IQGAP1 in PDAC. cytometry The interaction between ectopically expressed Flag-FBP1 and Cells were washed twice with PBS and resuspended in 1 Myc-IQGAP1 in 293T cells and endogenous FBP1 and IQGAP1 in 5 Binding Buffer. Cells (1 10 ) were stained with PE Annexin PANC-1 PDAC cells was confirmed by reciprocal coimmunopre- V and 7-amino-actinomycin following the manufacturer's instruc- cipitation (co-IP) assays (Fig. 1B). To define which region(s) in tions of PE Annexin V Apoptosis Detection Kit I (BD Biosciences). FBP1 mediate its interaction with IQGAP1, we constructed seven Cells were incubated for 15 minutes at room temperature and GST-FBP1 recombinant proteins corresponding to seven exons of analyzed on a flow cytometer. Data were analyzed with FlowJo FBP1 as reported previously (Fig. 1C; ref. 12). GST pull-down analysis software. assays demonstrated that GST-FBP1 E4 (amino acids 143–188), but not GST or other GST-FBP1 recombinant proteins, interacted Pancreatic cancer patient specimens, tissue microarray, IHC, with IQGAP1 (Fig. 1D). To determine which domain(s) of and staining scoring IQGAP1 are involved in FBP1 binding, we generated six GST- Pancreatic cancer tissue microarrays (TMA) were purchased IQGAP1 recombinant proteins corresponding to six well-charac- from US Biomax, Inc. (cat. # HPan-Ade150CS-01). TMA speci- terized functional domains of IQGAP1 (Fig. 1E). GST pull-down fi mens were immunostained with FBP1 and pERK1/2 antibodies as assays revealed that the WW domain of IQGAP1 speci cally described previously (14, 15). Staining intensity was graded/ interacted with FBP1 proteins expressed in PANC-1 cells (Fig. in vitro scored in a blinded fashion: 1 ¼ weak staining at 100 magni- 1F). Thus, as demonstrated by assays and in cultured PDAC fication but little or no staining at 40 magnification; 2 ¼ cells, FBP1 interacts with the scaffold protein IQGAP1. medium staining at 40 magnification; 3 ¼ strong staining at 40 magnification. A final staining index was calculated using the FBP1 competes with ERK1/2 to bind to the WW domain of formula: staining intensity percentage. IQGAP1 and inhibits ERK1/2 phosphorylation It is known that IQGAP1 interacts with c-RAF, MEK2, and ERK2 Generation of pancreatic cancer xenografts in mice and other proteins in mammalian cells (Fig. 2A; refs. 6–8, 18, 19). Six-week-old NOD/SCID IL2-receptor gamma null (NSG) mice Similar to these findings, we demonstrated that IQGAP1 inter- were generated in-house and used for animal experiments. The acted with components of the MAPK pathway in PANC-1 cells animal study was approved by the IACUC at Mayo Clinic. All mice (Fig. 2B). Importantly, we found that knockdown of FBP1 were housed in standard conditions with a 12-hour light/dark increased IQGAP1 interaction with both ERK1 and ERK2, but cycle and access to food and water ad libitum. MIA PaCa-2 cells had no overt effect on IQGAP1 interaction with c-RAF and MEK2 (5 106) infected with lentivirus [in 100 mL1 PBS plus 100 mL (Fig. 2B). ERK2 was known to bind to the WW domain of IQGAP1 Matrigel (BD Biosciences)] were injected subcutaneously into the (8). Our data also showed that FBP1 bound to the WW domain of right flank of mice. The volume of xenografts was measured every IQGAP1 (Figs. 1F and 2A). To determine the impact of FBP1 on other day for 21 days and calculated using the formula L W2 the interaction between IQGAP1 and ERK2, we generated His- 0.5. Upon the completion of measurement, tumor grafts were FBP1 and His-ERK2 constructs, purified these proteins from harvested. bacteria, and performed GST pull-down assays with GST or GST-IQGAP1 P3 (WW domain only). We demonstrated that FBP1 competed with ERK2 to bind to the WW domain of IQGAP1 in Statistical analysis vitro (Fig. 2C and D). All values were expressed as means SD. Statistical analyses Given that FBP1 inhibits IQGAP1–ERK interaction, we sought t P were performed with Student test unless otherwise indicated. to determine whether FBP1 regulates phosphorylation and acti- < fi values 0.05 are considered statistically signi cant. Pearson prod- vation of ERK1/2. We performed knockdown experiments in – uct moment correlation was used to calculate the correlation PANC-1 and MIA PaCa-2 PDAC cell lines. Knockdown of IQGAP1 between FBP1 and pERK1/2 staining index in PDAC TMAs. by two independent gene-specific shRNAs markedly decreased ERK1/2 phosphorylation (pERK1/2; Fig. 2E). Knocking down Additional methods FBP1 alone increased pERK1/2 (Fig. 2F). The effect of FBP1 Other detailed methods are described in Supplementary Mate- knockdown on ERK1/2 phosphorylation was completely reversed rials and Methods. by IQGAP1 knockdown (Fig. 2G). Conversely, overexpression of

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Figure 1. FBP1 binds to the scaffold protein IQGAP1. A, SDS-PAGE and silver staining of proteins purified by tandem affinity protein purification in 293T cells transiently transfected with SFB control vector or SFB-tagged FBP1. B, Western blot analysis of ectopically expressed Flag-FBP1 and Myc-IQGAP1 reciprocally immunoprecipitated by anti-Myc and anti-Flag in 293T cells, and endogenous FBP1 and IQGAP1 proteins reciprocally immunoprecipitated by anti-IQGAP1 and anti-FBP1 in PANC-1 cells. Immunoblots (IB) are representative of results from two independent experiments (n ¼ 2). C, Schematic diagram depicting a set of GST-FBP1 recombinant protein constructs. D, Western blot analysis of IQGAP1 proteins in PANC-1 whole cell lysate pulled down by GST or GST-FBP1 recombinant proteins. Immunoblots are representative of results from two independent experiments (n ¼ 2). Arrows, expected molecular weight. E, Schematic diagram depicting a set of GST-IQGAP1 recombinant protein constructs. F, Western blot analysis of FBP1 proteins in PANC-1 whole cell lysate pulled down by GST or GST-IQGAP1 recombinant proteins. Immunoblots are representative of results from two independent experiments (n ¼ 2). Arrows, expected molecular weight.

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IQGAP1 increased pERK1/2 in both PANC-1 and MIA PaCa-2 cell phorylation, we sought to determine whether expression of FBP1 lines, and this effect was largely diminished in cells cotransfected and pERK1/2 correlates in human PDAC specimens. We exam- with FBP1 (Fig. 2H). Next, we generated IQGAP1-depleted PANC- ined the expression of these two proteins by performing IHC on a 1 and MIA PaCa-2 cells (Fig. 2I; Supplementary Fig. S1A). TMA containing a cohort of pancreatic cancer samples (n ¼ 75 Although pERK1 was hardly detectable and pERK2 was very low normal-tumor paired TMA specimens) obtained from 75 patients. in IQGAP1-depleted cells, overexpression of FBP1 failed to affect IHC staining was evaluated by measuring both percentage of pERK1/2 in both cell lines (Fig. 2I). These data suggest that the positive cells and staining intensity. Representative images of low/ effect of FBP1 on pERK1/2 is mediated primarily through no and high staining of FBP1 and pERK1/2 and corresponding IQGAP1. Taken together, our results indicate that FBP1 competes hematoxylin and eosin staining are shown in Fig. 4A. PDAC with ERK1/2 to bind to the WW domain of IQGAP1 and inhibits tissues had lower expression of FBP1 (P ¼ 2.59e14) but higher ERK1/2 phosphorylation. pERK1/2 levels (P ¼ 1.02e05) compared with the adjacent normal tissues (Fig. 4B). Our analysis also showed that activation FBP1 inhibits ERK1/2 phosphorylation independent of its of pERK1/2 was mainly detected in higher tumor grade (G2 and enzymatic activity G3), and its expression positively correlated with tumor grades – ﬁ r ¼ To rule out the possibility that the enzymatic activity of FBP1 is (Pearson product moment correlation coef ciency P ¼ required to inhibit ERK1/2 phosphorylation, we constructed a 0.4043565, 0.0003207). In contrast, FBP1 expression inverse- – catalytically inactive mutant G260R of FBP1 as described previ- ly correlated with tumor grades (Pearson product moment cor- r ¼ P ¼ ously (20, 21). Reciprocal co-IP assays demonstrated that the relation 0.2674027, 0.02038; Fig. 4C and D). Further G260R mutation did not affect the IQGAP1–FBP1 interaction in analysis indicated that decreased expression of FBP1 correlated 293T cells (Fig. 3A and B). Ectopic expression of FBP1-WT and with increased levels of pERK1/2 in PDAC tissues of this cohort – ﬁ r ¼ FBP1-G260R mutant both decreased IQGAP1 interaction with (Pearson product moment correlation coef ciency 0.421, P ¼ ERK2, but not c-RAF and MEK2 (Fig. 3C). We transfected FBP1-WT 0.0125; Fig. 4E). These data indicate that loss of or reduced and G260R mutant into PANC-1 and MIA PaCa-2 cells and expression of FBP1 correlates with increased pERK1/2 levels, at demonstrated that ectopic expression of FBP1-WT and FBP1- least in a subset of PDAC patients, and deregulation of these G260R mutant resulted in similar inhibitory effect on pERK1/2 proteins associates with disease progression. in both cell lines (Fig. 3D). As expected, forced expression of FBP1- WT but not FBP1-G260R mutant inhibited glucose consumption A small FBP1-derived peptide inhibits IQGAP1 binding to and and lactate production in these cells (Fig. 3E and F). Our data phosphorylation of ERK1/2 indicate that FBP1 binds to IQGAP1 and inhibits ERK1/2 phos- GST pull-down assays indicated that GST-FBP1 E4 (143–188) phorylation in a manner independent of its enzymatic activity. interacted with IQGAP1 (Fig. 1D). We thus generated a GST- tagged bacterial expression vector for an FBP1 mutant in which the Decreased FBP1 expression correlates with increased pERK1/2 E4 domain is deleted (FBP1DE4; Fig. 5A). In vitro protein binding level in PDAC patient specimens studies demonstrated that GST-FBP1-WT, GST-FBP1-G260R It has been reported previously that FBP1 is downregulated in mutant, or GST-FBP1 E4 (143–188), but not GST or GST- human PDAC cell lines and patient samples. To explore clinical FBP1DE4 recombinant proteins, interacted with IQGAP1 (Fig. relevance of FBP1 inhibition of IQGAP1-mediated ERK1/2 phos- 5B). Furthermore, GST-IQGAP1 P3 (WW domain) was able to

Figure 2. FBP1 binds to the WW domain of IQGAP1 and inhibits ERK1/2 phosphorylation. A, Schematic diagram depicting the domain structure of IQGAP1 and previously defined proteins bound by each domain. B, Western blot analysis of whole cell lysate and co-IP samples in PANC-1 cells 48 hours after being infected with lentivirus expressing control shRNA or FBP1-specific shRNAs. MAPK pathway proteins (c-RAF, MEK2, ERK2, or ERK1) coimmunoprecipitated by IQGAP1 were quantified by ImageJ software and normalized to the quantified value of immunoprecipitated IQGAP1. The normalized values were further normalized to the value in cells infected with shControl. Immunoblots (IB) are representative of results from two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. C, His-FBP1 and His-ERK2 were purified from bacteria. His- ERK2 were incubated with different concentrations of His-FBP1 and subjected to GST pull-down using GST or GST-IQGAP1 P3 (FBP1-binding region in IQGAP1) and Western blot analysis using indicated antibodies. Western blots are representative of two independent experiments (n ¼ 2). Arrows, expected molecular weight. D, His-FBP1 were incubated with different concentrations of His-ERK2 and subjected to GST pull-down using GST or GST-IQGAP1 P3 and Western blot analysis using indicated antibodies. Data are representative of two independent experiments (n ¼ 2). Arrows, expected molecular weight. E, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 48 hours after being infected with lentivirus expressing control shRNA or IQGAP1-specific shRNA. pERK1, pERK2, and IQGAP1 proteins were quantified by ImageJ software and normalized to the quantified value of b-tubulin. The normalized values were further normalized to the value in cells infected with shControl. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. F, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 48 hours after being infected with lentivirus expressing control shRNA or IQGAP1-specific shRNA. pERK1, pERK2, and FBP1 proteins were quantified as in E. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. G, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 48 hours after being infected with lentivirus expressing control shRNA, FBP1-specific, IQGAP1-specific shRNA, or both shRNA. pERK2, FBP1, and IQGAP1 proteins were quantified as in E. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. H, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 24 hours after being transfected with indicated plasmids. pERK2 proteinswere quantified as in E . Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. I, PANC-1 and MIA PaCa-2 cells were infected with control and IQGAP1-specific shRNAs. After 24 hours, PANC-1 and MIA PaCa-2 cells were transfected with indicated constructs for another 24 hours, followed by Western blot analysis. Exp., exposure. pERK2and IQGAP1 proteins were quantified as in E. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments.

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FBP1 Inhibits IQGAP1 Activation of ERK

Figure 3. FBP1 inhibits ERK1/2 phosphorylation independent of its enzymatic activity. A and B, Western blot analysis of whole cell lysate and co-IP samples from PANC-1 cells 24 hours after being transfected with indicated plasmids. Myc-IQGAP1 in A were quantified and normalized to the quantified value of immunoprecipitated Flag-FBP1. Flag-FBP1 in B were quantified and normalized to the quantified value of immunoprecipitated Myc-IQGAP1.The normalized values were further normalized to the value in the cells transfected with Flag-FBP1 WT group. Data are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. C, Western blot analysis of whole cell lysate and co-IP samples in PANC-1 cells 24 hours after transfected with indicated plasmids. MAPK pathway proteins (c-RAF, MEK2, and ERK2) immunoprecipitated by IQGAP1 were quantified as in A.Western blots shown are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. D, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 24 hours after being transfected with indicated plasmids. pERK2 proteins were quantified and normalized to the quantified value of b-tubulin. The normalized values were further normalized to the shControl group. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. E and F, Measurement of glucose consumption and L-lactate production in the spent medium of PANC-1 and MIA PaCa-2 cells 48 hours after being transfected with indicated constructs. Data are shown as means SD (n ¼ 3). E.V., empty vector; n.s., not significant; , P < 0.01. pull down SFB-FBP1-WT, SFB-FBP1-G260R mutant and SFB-FBP1 SFB-FBP1DE4 decreased IQGAP1 interaction with ERK2, but not E4, but not SFB-FBP1DE4 (Fig. 5C). These data revealed that the E4 c-RAF and MEK2 (Fig. 5D). The SFB empty vector (SFB-EV) and fragment of FBP1 is required for its interaction with the WW SFB-FBP1-WT were included as a negative and positive control, domain of IQGAP1 in vitro. respectively. We also demonstrated that expression of SFB-FBP1 Next, we sought to determine whether FBP1 E4 alone is able to E4 but not SFB-FBP1DE4 inhibited pERK1/2 in both PANC-1 and inhibit IQGAP1–ERK protein interaction and regulates pERK1/2. MIA PaCa-2 cell lines (Fig. 5E). We further showed that IQGAP1- We demonstrated that ectopic expression of SFB-FBP1 E4 but not enhanced pERK1/2 was attenuated by forced expression of

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Figure 4. Expressions of FBP1 and pERK1/2 inversely correlate in PDAC patient specimens. A, Representative images of IHC of anti-FBP1 and anti-pERK1/2 antibodies and hematoxylin and eosin (H&E) staining of TMA (n ¼ 75) tissue sections. Scale bars are shown as indicated. B, Box plots of FBP1 and pERK1/2 expression as indicated by the staining index (SI) in paired normal-tumor tissues from PDAC patients (n ¼ 75). The P values are also shown. C, Box plots of FBP1 and pERK1/2 expression determined as indicated by the staining index at different tumor grades. G1, G2, and G3 represent well-differentiated, moderately differentiated, and poorly differentiated tumors, respectively. The P values are also shown. D, Waterfall diagram showing staining index of FBP1 and pERK1/2 in TMA and association with tumor grades. E, Correlation analysis of the staining index for expression of FBP1 and pERK1/2 proteins in PDAC patient specimens (n ¼ 75). Pearson product-moment correlation coefﬁciency and the P values are also shown.

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Figure 5. A small FBP1-derived peptide inhibits IQGAP1 binding to ERK proteins and ERK1/2 phosphorylation. A, Schematic diagram depicting a set of GST-FBP1 recombinant protein constructs. B, Western blot analysis of IQGAP1 proteins in PANC-1 whole cell lysate pulled down by GST or GST-FBP1 recombinant proteins. Data are representative of two independent experiments (n ¼ 2). C, Western blot analysis of SFB-tagged recombinant proteins in PANC-1 whole cell lysate pulled down by GST or GST-IQGAP1-P3 recombinant proteins. Western blots are representative of two independent experiments (n ¼ 2). D, Western blot analysis of whole cell lysate and co-IP samples from PANC-1 cells 24 hours after being transfected with indicated plasmids. MAPK pathway proteins (c-RAF, MEK2, and ERK2) coimmunoprecipitated by IQGAP1 were quantified and normalized to the quantified value of immunoprecipitated IQGAP1. The normalized values were further normalized to the value in cells transfected with SFB-EV. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. E, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 24 hours transfected with indicated plasmids. pERK2 proteins were quantified as in D. Western blots are representative of results from two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. F, Western blot analysis of whole cell lysate of PANC-1 cells 24 hours after being transfected with indicated plasmids. pERK2 was quantified as in D. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. G and H, Measurement of glucose consumption and L-lactate production in the spent medium of PANC-1 and MIA PaCa-2 cells 48 hours after transfection with indicated constructs. Data are shown as means SD (n ¼ 3). n.s., not significant; , P < 0.01.

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Figure 6. A small FBP1-derived peptide inhibitor suppresses pancreatic cancer growth in culture and in mice. A and B, PANC-1 and MIA PaCa-2 cells were infected with lentivirus expressing control shRNA or FBP1-specific shRNA. Twenty-four hours after infection, control shRNA and FBP1 shRNA-infected cells were further infected with lentiviral vectors as indicated. Forty-eight hours after puromycin selection, cells were harvested for Western blots (A) and MTS assay (B). pERK2 proteins were quantified and normalized to the quantified value of b-tubulin. The normalized values were further normalized to the control group as shown in the panel. Western blots in A are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. Data shown in B are mean values SD from six replicates (n ¼ 6). , P < 0.01. C and D, MIA PaCa-2 cells were infected with lentivirus as in A, and 72 hours after infection and puromycin selection, cells were injected subcutaneously into the right flank of NSG mice and tumor growth was measured for 21 days. Tumors in each group at day 21 were harvested, photographed, and are shown in C. Data in D are shown as means SD (n ¼ 5). , P < 0.01 comparing size of tumors in different groups at day 21.

SFB-FBP1 E4 but not SFB-FBP1DE4 in PANC-1 cells (Fig. 5F). sion (22–24). We sought to investigate the role of FBP1 in Moreover, in agreement with the fact that FBP1 E4 did not contain pancreatic cancer cell proliferation. PANC-1 and MIA PaCa-2 cells the enzymatic domain of FBP1, it is not surprising that FBP1 E4 were infected with control shRNA or FBP1-specific shRNA. Cells had no effect on glucose metabolism in both PDAC cell lines infected with FBP1-specific shRNAs were further transfected with examined (Fig. 5G and H). These findings further imply that FBP1 empty lentiviral expression vector (pTsin) or shRNA-resistant WT inhibition of ERK1/2 phosphorylation is not only exerted in a FBP1 (pTsin-SFB-FBP1SR) or two mutants (pTsin-SFB-FBP1- fashion independent of its enzymatic activity, but also uncoupled G260RSR or pTsin-SFB FBP1 E4). These cells were used for Western from glucose consumption and lactate production. Taken togeth- blot analysis, MTS assay, and animal studies. As demonstrated er, these data indicate that a small FBP1-derived peptide (FBP1 in Fig. 6A, both FBP1 knockdown and SFB-FBP1, SFB-FBP1 E4) is able to inhibit IQGAP1 binding to ERK proteins and ERK1/2 G260R, SFB-FBP1 E4 overexpression were effective in both phosphorylation. PANC-1 and MIA PaCa-2 cells. The corresponding changes of pERK1/2 were also detected in cells infected with different vectors A small FBP1-derived peptide inhibitor suppresses PDAC cell (Fig. 6A). MTS assay indicated that knockdown of FBP1 alone growth in culture and in mice increased cell proliferation compared with the control group (Fig. Previous studies report that the RAS–RAF–MEK–ERK signaling 6B). Notably, restored expression of shRNA-resistant FBP1-WT, pathway promotes pancreatic cancer proliferation and progres- FBP1-G260R, or FBP1 E4 invariably inhibited cell proliferation. It

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is not surprising that the growth-inhibitory ability of FBP1-G260R ERK2 (Supplementary Fig. S2C) and the cell-cycle effects of or FBP1 E4 was not as potent as FBP1-WT (Fig. 6B) because the gemcitabine (Supplementary Fig. S2D–S2F). Notably, knock- enzymatic activity in FBP1-WT may also contribute to growth down of IQGAP1 abolished gemcitabine-induced pERK1/2 in inhibition. Similar to the results in MIA PaCa-2 cells in culture, both cell lines (Fig. 7A). We transfected PANC-1 and MIA PaCa-2 knockdown of FBP1 alone promoted growth of MIA PaCa-2 cell lines with control or FBP1 E4 expression vectors and treated xenografts in mice (Fig. 6C and D). However, this effect was cells with DMSO or gemcitabine. FBP1 E4 expression impeded reversed by restored expression of shRNA-resistant FBP1-WT, gemcitabine-induced ERK activation in both cell lines (Fig. 7B). FBP1-G260R, or FBP1 E4 (Fig. 6C and D). These data suggest MTS assays demonstrated that gemcitabine treatment combined that FBP1 and a small FBP1-derived peptide (FBP1 E4) are able to with FBP1 E4 expression resulted in greater reduction of cell suppress PDAC cell growth in culture and in mice. proliferation than gemcitabine alone (Fig. 7C). Moreover, cotreatment of cells with gemcitabine and FBP1 E4 induced higher cell The FBP1-derived peptide impedes gemcitabine-induced ERK death than gemcitabine alone (Fig. 7D). Collectively, these data activation and chemoresistance suggest that the FBP1-derived small peptide impedes gemcita- The nucleoside analogue gemcitabine is a leading therapeutic bine-induced ERK activation and overcomes chemoresistance in agent for pancreatic cancer (25). However, it fails to significantly PDAC cells. improve the outcome of pancreatic carcinoma patients due to acquisition of chemoresistance in most tumor cells (26). It is well documented that gemcitabine treatment results in activation of Discussion the RAS–RAF–MAPK pathway (27). However, the mechanism IQGAP proteins are an evolutionarily conserved multistruc- underlying gemcitabine-induced MAPK activation remains poor- tural domain protein family, which participate in cell adhesion, ly understood. Moreover, gemcitabine sensitivity can be increased migration, signaling, division, and many other biological pro- through combination with a pERK-reducing agent (28). We cesses (32, 33). The IQGAP family is comprised of three members sought to determine whether FBP1 E4 can restore gemcitabine in humans. IQGAP1, which is the most characterized, is upregu- sensitivity in PDAC cells through inhibition of pERK1/2. In lated in several tumor types, including pancreatic carcinoma, agreement with the findings in the BXPC-3 PDAC cell line (27) gastric carcinoma, and hepatocellular carcinoma. IQGAP1 can and RAS mutation–induced mouse PDAC tumors (29), we found bind to Cdc42 and Rac1, E-cadherin, b-catenin, calmodulin, and that the mean value of pERK1/2 staining was relatively higher in components of the MAPK pathway through protein–protein gemcitabine-treated PDAC patient specimens compared with that interaction and plays an important role in tumorigenesis (17). in untreated counterparts (Supplementary Fig. S1B and S1C). It is IQGAP1 is a MAPK scaffold essential for effective propagation of worth noting that the difference in pERK1/2 staining between the MAPK signaling cascade (6). Our data demonstrate that the these two groups was not statistically significant, which could be tumor suppressor protein FBP1 inhibits PDAC cell growth by due to the very small number of gemcitabine-treated PDAC binding with IQGAP1 and impeding IQGAP1-dependent ERK1/2 patient samples (Supplementary Fig. S1C). phosphorylation and activation. Therefore, in tumors where Similar to the previous reports (30, 31), we found that MIA IQGAP1 is overexpressed (34–36), overexpression of IQGAP1 PaCa-2 cells are gemcitabine sensitive and PANC-1 cells are may represent an alternative to promote cancer progression by gemcitabine resistant (Supplementary Fig. S2A). In agreement overcoming the inhibitory effect of FBP1 expression on ERK1/2 with these findings, we demonstrated that gemcitabine-sensitive activity. MIA PaCa-2 cells have much higher FBP1 expression and much The MAPK pathway is important for cancer cell proliferation, lower pERK1/2 compared with gemcitabine-resistant PANC-1 migration, survival, and resistance to cancer therapy (37). A cells (Supplementary Fig. S2B). Moreover, we demonstrated that variety of MAPK blockers, including MEK inhibitors and ERK gemcitabine treatment increased pERK1/2 levels in both PANC-1 inhibitors, has been developed (38). These agents are relatively and MIA PaCa-2 cell lines (Fig. 7A and B), which is consistent with ineffective in many clinical trials (39). Combinations of MAPK the effects of gemcitabine in PDAC tumors in mice and patients blockers with cytotoxic chemotherapy and/or other targeted (Supplementary Fig. S1B and S1C; ref. 29). We further showed agents are being examined to expand the efficacy of cancer that gemcitabine-induced elevation of pERK1/2 was unlikely due therapies. Also, scaffold–kinase interaction blockade, a novel to the effect of gemcitabine on IQGAP1 interaction with ERK1 and mechanism different from direct kinase inhibition, represents a

Figure 7. FBP1 E4 peptide impedes gemcitabine-induced ERK activation and chemoresistance. A, Western blot analysis of whole cell lysate of PANC-1 and MIA PaCa-2 cells 48 hours after infection with lentivirus expressing indicated shRNAs. PANC-1 cells were treated with or without gemcitabine (10 mmol/L) and MIA PaCa-2 cells with or without gemcitabine (5 mmol/L) for 24 hours prior to harvest. pERK2 proteins were quantified and normalized to the quantified value of b-tubulin. The normalized values were further normalized to the shControl group. Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. B–D, PANC-1 and MIA PaCa-2 cells were transfected with indicated plasmids. Twenty-four hours after transfection, PANC-1 cells were treated with or without gemcitabine (10 mmol/L) and MIA PaCa-2 cells with or without gemcitabine (1 mmol/L). Forty-eight hours after transfection, cells were harvested for Western blot (B), MTS assay (C), and Annexin V assay and flow cytometry analysis (D). pERK2 in B was quantified as in A.Western blots are representative of two independent experiments (n ¼ 2), and the quantified value shown was the average of the normalized values for each protein band from the two independent experiments. For panel C, data shown are mean values SD from six replicates (n ¼ 6). , P < 0.01 for comparison at 48 hours. Original data of FACS analysis (left) and quantified results from two biological replicates (right) are shown in D. E and F, Models depicting that FBP1 inhibits ERK activation and gemcitabine resistance in pancreatic cancer by blocking IQGAP1–MAPK kinase interaction. In pancreatic cancer cells, IQGAP1 functions as a scaffold for activation of RAF, MEK, and ERK. Loss of FBP1 promotes aberrant ERK activation and gemcitabine resistance (E). A small FBP1-derived peptide competes with ERK to bind to the WW domain of IQGAP1 and inhibits ERK1/2 phosphorylation, thereby resensitizing pancreatic cancer cells to the treatment of gemcitabine (F).

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FBP1 Inhibits IQGAP1 Activation of ERK

promising strategy to improve the efficacy of cancer treatments derived peptide inhibitor. Most importantly, we define a bioactive (8). Aberrant activation of the RAS–RAF–MEK–ERK1/2 pathway FBP1-derived peptide inhibitor, which inhibits gemcitabine- occurs in more than 90% of human pancreatic cancers. Our induced ERK activation and enhances the efficacy of gemcitabine present study demonstrates that ERK activation can be abolished in PDAC (Fig. 7E and F). These findings highlight that inhibition by genetic depletion of IQGAP1 or treating cells with a small of the IQGAP1–ERK1/2 signaling axis by FBP1 can be harnessed FBP1-derived peptide inhibitor. Thus, FBP1-mediated suppres- for effective treatment of PDAC. sion of the IQGAP1–MAPK interaction may be a new target of pancreatic cancer therapy. Disclosure of Potential Conflicts of Interest Gemcitabine has emerged as the leading chemotherapeutic A patent application (CN 105734031 A) has been submitted by Xin Jin and agent for the treatment of PDAC. However, gemcitabine fails to Haojie Huang based on the results reported in this study. No potential conflicts significantly improve the outcome of pancreatic carcinoma of interest were disclosed by the other authors. patients due to development of chemoresistance in patients. Gemcitabine treatment often induces ERK activation (28), which Authors' Contributions has been recognized as a cause of drug resistance to pancreatic fi Conception and design: X. Jin, H. Huang cancer therapies. Our ndings show that increased ERK1/2 phos- Acquisition of data (provided animals, acquired and managed patients, phorylation induced by gemcitabine treatment can be inhibited provided facilities, etc.): L. Zhang, A.H. Tang, H. Wu by cotreatment of cells with IQGAP1 knockdown or a small FBP1- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, derived peptide inhibitor. Thus, the IQGAP1–MAPK interaction computational analysis): X. Jin, L. Wang, T. Ma, L. Zhang may represent a key mechanism responsible for ERK activation Writing, review, and/or revision of the manuscript: X. Jin, H. Wu, H. Huang and gemcitabine resistance in PDAC. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Jin, A.H. Tang, D.D. Billadeau FBP1 is a rate-limiting enzyme in gluconeogenesis and func- Study supervision: H. Wu, H. Huang tions as a tumor suppressor in numerous cancer types, including Other (technical support): Y. Pan pancreatic cancer. Decreased expression of FBP1 associates with advanced tumor stage and poor overall survival in PDAC patients Acknowledgments (11). Ectopic expression of FBP1 inhibits tumor growth in renal The authors thank W.R. Bamlet and J.J. Brooks at Mayo Clinic for acquisition carcinoma and hepatocellular carcinoma (10, 12). It has been of pERK1/2 staining data from gemcitabine-treated and untreated PDAC patient documented that FBP1 suppresses tumor progression mainly samples. through reducing aerobic glycolysis to inhibit the Warburg effect (10), or by inhibiting nuclear HIF function in renal carcinoma Grant Support (12). Our data show that FBP1 inhibits tumor growth in PDAC. This work was supported in part by grants from the NIH (CA134514, Mechanistically, we further show that FBP1 binds to the WW – CA130908, and CA193239 to H. Huang and CA140550 to A.H. Tang), the domain of IQGAP1 and disrupts the interaction of IQGAP1 Pancreatic Cancer SPORE grant (P50 CA102701 to D.D. Billadeau), and ERK1/2 in a manner independent of FBP1 enzymatic activity, Scientific Research Training Program for Young Talents of Union Hospital, representing a previously uncharacterized role of FBP1 in inhi- Tongji Medical College, Huazhong University of Science and Technology, bition of tumor growth and progression. Wuhan, China. Research supported by the 2010 Pancreatic Cancer Action In summary, we demonstrate that FBP1 acts as a negative Network-AACR Innovative Grant, Grant Number 10-60-25-TANG (A.H. Tang). The costs of publication of this article were defrayed in part by the payment of modular of the IQGAP1–MAPK signaling axis by binding to the page charges. This article must therefore be hereby marked advertisement in WW domain of IQGAP1 and impeding IQGAP1-dependent accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ERK1/2 phosphorylation, independent of its enzymatic activity, in PDAC cells. We further show that ERK activation is abolished by Received November 17, 2016; revised April 4, 2017; accepted June 16, 2017; genetic depletion of IQGAP1 or treating cells with a small FBP1- published OnlineFirst July 18, 2017.

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OF14 Cancer Res; 77(16) August 15, 2017 Cancer Research

Fructose-1,6-bisphosphatase Inhibits ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking IQGAP1−MAPK Interaction

Xin Jin, Yunqian Pan, Liguo Wang, et al.

Cancer Res Published OnlineFirst July 18, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-3143

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