Research Article GATA1 Promotes Gemcitabine Resistance in Pancreatic Cancer Through Antiapoptotic Pathway

Hindawi Journal of Oncology Volume 2019, Article ID 9474273, 16 pages https://doi.org/10.1155/2019/9474273

Research Article GATA1 Promotes Gemcitabine Resistance in Pancreatic Cancer through Antiapoptotic Pathway

Zhenyu Chang,1 Yanan Zhang,2 Jie Liu,2 Chengjian Guan,1 Xinjin Gu,1 Zelong Yang,1 Qinong Ye ,2 Lihua Ding ,2 and Rong Liu 1

1 Department of Hepatobiliary and Pancreatic Surgical Oncology, Medical School of Chinese People’s Liberation Army, Beijing 100853, China 2Department of Medical Molecular Biology, Beijing Institute of Biotechnology, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China

Correspondence should be addressed to Qinong Ye; [email protected], Lihua Ding; [email protected], and Rong Liu; [email protected]

Received 25 December 2018; Revised 27 February 2019; Accepted 28 February 2019; Published 10 April 2019

Academic Editor: Tomas E. Adrian

Copyright © 2019 Zhenyu Chang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gemcitabine-based chemotherapy is the frst-line treatment for pancreatic cancer. However, chemoresistance is a major obstacle to drug efcacy, leading to poor prognosis. Little progress has been achieved although multiple mechanisms are investigated. Terefore, efective strategies are urgently needed to overcome drug resistance. Here, we demonstrate that the transcription factor GATA binding protein 1 (GATA1) promotes gemcitabine resistance in pancreatic cancer through antiapoptotic pathway. GATA1 is highly expressed in pancreatic ductal adenocarcinoma (PDAC) tissues, and GATA1 status is an independent predictor of prognosis and response to gemcitabine therapy. Further investigation demonstrates GATA1 is involved in both intrinsic and acquired gemcitabine resistance in PDAC cells. Mechanistically, we fnd that GATA1 upregulates Bcl-XL expression by binding to its promoter and thus induces gemcitabine resistance through enhancing Bcl-XL mediated antiapoptosis in vitro and in vivo.Moreover, in PDAC patients, Bcl-XL expression is positively correlated with GATA1 level and predicts clinical outcomes and gemcitabine response. Taken together, our results indicate that GATA1 is a novel marker and potential target for pancreatic cancer. Targeting GATA1 combined with Bcl-XL may be a promising strategy to enhance gemcitabine response.

1. Introduction pathways, resistance to apoptosis, and change of tumor and stromal microenvironment [6]. Particularly, recent studies Pancreatic ductal adenocarcinoma (PDAC) is one of the most demonstrated that signaling pathways modulating prolifer- aggressive tumors with poor prognosis [1]. Te 6-month ation, diferentiation, apoptosis, invasion, and angiogenesis, recurrence-free survival (RFS) of PDAC remains below 15%, including MAPK, PI3K/Akt, and NF-�B, directly or indi- and the overall survival (OS) rate at 5 years less than 8% rectly regulate gemcitabine sensitivity in PDAC cells [7– [2]. Te dismal prognosis is largely attributed to extreme 10]. However, mechanisms related to gemcitabine resistance chemoresistant phenotype of the tumor [3]. At present, are not well elucidated. Further understanding of pathways gemcitabine remains a standard chemotherapeutic agent mediating gemcitabine chemoresistance is crucial for devel- for advanced pancreatic cancer and postsurgery adjuvant oping improved treatments and prolonging patients’ survival therapy [4]. However, most patients developed resistance in pancreatic cancer. within weeks of gemcitabine treatment, and under 25% of Te major action mode of gemcitabine is to induce apop- patients with PDAC benefted from gemcitabine treatment tosis of cancer cells [11]. Te profound deregulation of the [5]. apoptotic machinery is one of the central events during devel- Te mechanisms of gemcitabine resistance include failure opment of chemoresistance in cancer cells. Gemcitabine- of drug uptake and metabolism, activation of DNA repair induced apoptosis involves the mitochondria-mediated 2 Journal of Oncology signaling pathway, which is mainly modulated by B-cell Bcl-XL cDNA were inserted into PSIH-H1-puro (System lymphoma-2 (Bcl-2) family proteins [12]. Te antiapoptotic Biosciences). Te small interfering RNAs sharing the same Bcl-2 family proteins include Bcl-2, Bcl-XL, Bcl-w,Mcl-1, and targets with shRNAs were synthetized from GenePharma BCL2A1 [13]. Bcl-XL is well known as a major antiapoptotic (Shanghai, China). Te sequences for siRNAs and shRNAs protein in pancreatic cancer. It is frequently upregulated in are listed in Table S4a. chemoresistant cells, counteracting the function of proap- Specifc antibodies against GATA1 (ab28839) and Bax optotic proteins [14]. Te knockdown of Bcl-XL signifcantly (ab32503) were purchased from Abcam. Anti-Bcl-XL sensitized pancreatic cancer cells to gemcitabine-mediated (#2764), anti-cleaved PARP (#5625), anti-cleaved caspase 3 apoptosis [15]. Multiple pathways are involved in gemcitabine (#9664), and anti-cleaved caspase 9 (#9505) were from Cell resistance through targeting Bcl-XL. For instance, MAPK Signaling Technology. Anti-�-actin (sc-47778HRP) was from and HIF-1� signaling upregulates Bcl-XL level and thus Santa Cruz Biotechnology. Gemcitabine (T0251) was ob- induces gemcitabine resistance in PDAC cells [16–19]. tained from Targetmol. Activated Akt favors cell survival via the direct regulation of Bcl-XL [20, 21]. However, the regulatory network of Bcl-XL 2.2. Clinical Samples. 86 pairs of PDAC tissues and adjacent expression is still not well clarifed. normal pancreas tissues and 86 separate PDAC tissues were GATA1 is the founding member of GATA transcription obtained from Chinese PLA General Hospital. In the 86 pairs factor protein family, which contains two highly conserved of cases, 59 of them received gemcitabine treatment, and zinc fnger domains, N-terminal fnger (NF), and C-terminal the other 27 pairs were chemonaive. All patients received fnger (CF). GATA1 NF has been reported to bind inde- radical surgery and were diagnosed by pathologic evidence pendently to GAT(C/G) sequences, and GATA1 CF binds from January 2011 to May 2017. Te mean follow-up time with high afnity and specifcity to (A/T)GATA(A/G) motifs is 23.0 months (1.1-86.6 months). Of 172 cases, 119 received [22]. GATA1 regulates its target genes through binding to adjuvant gemcitabine-based chemotherapy and 53 did not consensus DNA sequence. GATA1 was frst found critical for receive any chemotherapy. For Western blot analysis, another the formation of early eosinophil precursors and for difer- 30 pairs of fresh frozen PDAC and adjacent normal tissues entiation of committed erythroid precursors and megakary- were collected. All samples were obtained with the informed ocytes [23]. Recently, GATA1 was reported to be involved consent of patients and with the approval of the Institutional in cell growth, apoptosis, tumorigenesis, and aggressiveness Review Committees of Chinese PLA General Hospital. of solid tumors. In breast cancer, GATA1 is overexpressed and promotes survivin expression [24]. Furthermore, the 2.3. Cell Culture and Transfection. Human embryonic kid- interaction of GATA1 and MMP-2 enhanced glioblastoma ney cell line HEK 293T, human normal pancreatic duct invasion and migration [25]. We have previously demon- epithelial cell line HPDE6c7, and human PDAC cell lines strated that GATA1 promotes breast cancer growth and (AsPC-1, BxPC-3, CFPAC-1, PANC-1, and SW1990) were metastasis through regulating VEGF expression [26], but the purchased from American Type Culture Collection (ATCC) role of GATA1 in PDAC remains unexplored. andculturedincorrespondingmediums(Gibco)suggested In this study, we found that GATA1 was upregulated in by ATCC. PDAC patients and correlated with RFS and OS, specifcally Transient transfection of plasmids was conducted using in patients treated with gemcitabine. Terefore, we further Lipofectamine 2000 Reagent according to the manufacturer’s investigated the function of GATA1 in gemcitabine resistance recommendations (Invitrogen). Transient transfections of andconfrmedthatGATA1inducedintrinsicandacquired siRNAs were performed using RNAimax Reagent according gemcitabine resistance through regulating Bcl-XL in vivo and to the manufacturer’s recommendations (Invitrogen). Stable in vitro. Tus, GATA1 inhibition alone or in combination with cell lines were obtained by lentiviral transduction with pCDH Bcl-XL inhibition may be a useful strategy for the treatment of or PSIH-H1-puro. Lentiviruses were obtained by cotransfect- gemcitabine-resistant PDAC patients overexpressing GATA1. ing recombinant lentivirus vectors and pPACK Packaging Plasmid Mix (System Biosciences) into HEK 293T cells 2. Methods with Megatran reagent (Origene). Viral supernatants were collected and fltered 48 hours afer transfection and sub- 2.1. Plasmids, Lentiviruses, siRNAs, and Reagents. Te sequently added to the culture medium of target cells with eukaryotic expression constructs for GATA1 were generated 8 �g/ml polybrene (Sigma-Aldrich). Te target cells were by cloning PCR-amplifed full length sequences into selected for 30 days with 1 �g/ml puromycin 48 h afer pcDNA3 (Invitrogen). GST-fusion protein encoding vectors infection to generate stably cell lines. were constructed by inserting PCR-amplifed sequences into pGEX-KG (Amersham Pharmacia Biotech). Te 2.4. Generation of Gemcitabine-Resistant Cells. PANC-1 and Bcl-XL promoter luciferase reporters were obtained by CFPAC-1 cells were cultured in medium containing gradually cloning PCR-amplifed promoter fragments into pGL4-basic increasing doses of gemcitabine ranging 1-50 �Mand5- vector (Promega). Te mutated Bcl-XL promoter luciferase 150 �M for PANC-1 and CFPAC-1 cells, respectively. Afer reporters were constructed by recombinant PCR. Lentiviral over 3 months of selection, cells were stably passaged in vector of GATA1 was constructed by cloning PCR-amplifed medium with low concentration of gemcitabine (1 �Mand full length sequences into pCDH (System Biosciences). 5 �M respectively) for more than 10 generations. Te resis- Te short hairpin RNAs (shRNAs) targeting GATA1 and tance status was confrmed by CCK8 assay. Journal of Oncology 3

2.5. Western Blot Analysis. Cells and tissues were lysed in amplifed by qRT-PCR to determine relative enrichment. RIPA lysis bufer containing protease inhibitors for 30 min. Primer sequences used were listed in Table S4c. Tissues were grinded into homogenates before lysis. Equal amounts of protein were separated by 10% or 15% SDS-PAGE 2.11. Flow Cytometry Assay. Cell apoptosis was analyzed and transferred to NC membranes. Afer blocking for 1 h, usinganAnnexinV-APC/PIapoptosisassaykit(KeyGEN the membranes were incubated with indicated antibodies and BioTECH). Afer being treated with or without gemcitabine detected by enhanced chemiluminescence. for 48 h, cells were digested, washed, resuspended, and stained with Annexin V-APC and PI according to the man- 2.6. Cell Proliferation and Cell Viability Assay. For cell prolif- ufacture’s protocol. Te apoptotic cells were detected with a eration assay, approximately 3000 cells per well were plated fow cytometer (BD Biosciences). ∘ into 96-well plates and incubated overnight at 37 C. Cell numbers was assessed by CCK-8 Kit (Dojindo Laboratories) 2.12. Animal Experiments. Protocols were approved by the following the manufacturer’s protocols afer incubating for Institutional Animal Care and Use Committee of Beijing 7 0, 24, 48, 72, or 96 h. Te absorbance at 450 nm of each Institute of Biotechnology. 1×10 PANC-1 cells stably infected well was examined by a microplate reader. For cell viability 4 with indicated lentiviruses were subcutaneously injected into assay, approximately 1×10 cells per well were plated into 96- ∘ the lef oxter of 7-week-old BABL/c nude mice. Two weeks well overnight at 37 C. Ten cells were treated with diferent afer implantation, gemcitabine (50 mg/kg) or control was concentrations of gemcitabine for 48 h before CCK8 assay. given intraperitoneally twice a week for 30 days. Tumor IC50 values were generated by an online IC50 calculator growth was monitored by vernier caliper measurement every (http://www.aatbio.com/tools/ic50-calculator/). 3 days and the tumor volume was calculated according to the following formula: volume = (longest diameter × 2 2.7. Colony Formation Assay. Approximately 1000 cells per shortest diameter )/2. Mice were sacrifced on day 30 afer well were seeded into 6-well plates and cultured in regular ∘ treatment. Tumors were excised and parafn-embedded. Te medium at 37 C. For gemcitabine resistance experiment, cells tissue sections were used in immunohistochemistry to detect were treated with gemcitabine (1 �Mand5�M for CFPAC- GATA1 and Bcl-XL expression. TUNEL assay was performed 1 and PANC-1 respectively) before plating. Afer 10-14 days, using one-step TRITC-labeled TUNEL assay kit (KeyGEN cells were fxed in 4% paraformaldehyde for 10 min and BioTECH). Parafn-embedded tissue sections were deparaf- stained with 0.1% crystal violet solution for 20 min. Images fnized, rehydrated, incubated with Proteinase K, reacted with were scanned and colony numbers were quantifed. TdT enzyme, and labeled with TRITC and DAPI according to the manufacture’s protocol. Fluorescence images of stained 2.8. Quantitative Reverse Transcription-PCR (qRT-PCR). tissue sections were collected under a laser confocal micro- Total RNA was extracted using RNAzol regent (Sigma) fol- scope. TUNEL-positive cells were detected and quantifed to lowing the manufacture’s protocol. Equal amount of RNA was determine the apoptotic index. reverse-transcribed using SuperScript II Reverse Transcrip- tase (Invitrogen). Real-time quantitative PCR was performed 2.13. Immunohistochemistry. Immunohistochemistry (IHC) with SYBR-green premix (Takara) on a CFX96 Real-Time of formalin-fxed parafn-embedded samples was performed PCR detection system. Te relative fold change of target −ûûCt as described previously [27]. Rabbit anti-GATA1 and rabbit mRNAs was normalized to �-actin calculated by 2 anti-Bcl-XL were used at dilutions of 1:1000 as primary method. Primer sequences used were listed in Table S4b. antibodies for IHC. Te expression of GATA1 and Bcl-XL was determined by H score method. H score was generated by 2.9. Dual-Luciferase Reporter Assay. Cells were seeded into multiplying the percentage of stained cells (0-100%) by the 24-well plates at 40∼60% confuency and cotransfected with intensity of the staining (low, 1+; medium, 2+; strong, 3+). diferent luciferase constructs and indicated expression vec- Tus, the score is between 0 and 3. Te median score was used tors or siRNAs and Renilla luciferase plasmid using Lipofec- to categorize low and high expression groups. We defned tamine 2000 Reagent. 48 h afer transfection, the cells were ≤1.215 as low GATA1 and ≤1.815 as low Bcl-XL. harvested, lysed and analyzed for luciferase activity with dual- luciferase assay kit (Vigorous) according to the manufacture’s 2.14. Statistical Analysis. Diferences between groups were protocol. Firefy luciferase activity was normalized to Renilla compared using student’s t test if a normal distribution is luciferase activity as control of transfection efciency. Rela- satisfed; otherwise, the nonparametric Mann-Whitney U test tive luciferase activity was assayed by the luminometer. was applied. For multiple comparisons, one-way ANOVA was adopted. Repeated measures ANOVA were used to 2.10. Chromatin Immunoprecipitation (ChIP) Assay. ChIP compare cell proliferation and viability curves and tumor assay was conducted with the Magna ChIP kit as manual growth curves. For survival and recurrence analysis, Kaplan- described (Millipore). Briefy, CFPAC-1 cells were cross- Meiermethodwasconductedwiththelog-ranktest.Te linked with freshly prepared 1% formaldehyde. Cell lysis, Cox regression model was used to perform univariate and sonicating, dilution, immunoprecipitation, washing, and multivariate analyses. Te correlation between GATA1 and elution were subsequently performed following the man- Bcl-XL scores was verifed by Spearman rank correlation ufacture’s protocol. Te purifed DNA samples were then analysis. By the correlation between clinical characteristics 4 Journal of Oncology and GATA1, Bcl-XL expression was evaluated by Fisher exact GATA1 knockdown reduced cell proliferation, and GATA1 test. All statistical analyses were performed using SPSS 23.0. reexpression in the knockdown cells rescued this efect All statistical tests were two-sided and P value <0.05 was (Figures 2(b) and S2a). Consistent with the cell proliferation considered statistically signifcant. results, GATA1 overexpression in CFPAC-1 and PANC-1 cells increased cell colony formation. GATA1 knockdown 3. Results decreased cell colony formation, and reexpression of GATA1 abolished this efect (Figures 2(c) and S2b). Aferwards, we 3.1. GATA1 Is Highly Expressed in PDAC. To explore the investigated the efect of GATA1 on gemcitabine resistance clinical signifcance of GATA1 in PDAC, we performed in pancreatic cancer cells. Compared to control cells (IC50 immunohistochemistry (IHC) to determine GATA1 protein value, CFPAC-1: 6.33 �M; PANC-1: 37.88 �M), GATA1 knock- expression in 86 pairs of PDAC and matched paracancerous down decreased the IC50 value of CFPAC-1 (IC50 value: tissues. GATA1 antibody specifcity was confrmed with anti- 1.61 �M) and PANC-1 cells (IC50 value: 9.72 �M), and the gen competition and Western blot of lysates from CFPAC-1 efect was abolished by GATA1 reexpression (IC50 value, and PANC-1 cells stably expressed GATA1 shRNA (Figures CFPAC-1: 6.25 �M, PANC-1: 36.77 �M), suggesting GATA1 S1a and S1b). Compared with paracancerous tissue, GATA1 knockdown increased sensitivity to gemcitabine, and this expression was upregulated in PDAC tissues (P =0.0023) phenotype was reversed by GATA1 reexpression (Figures 2(d) (Figures 1(a) and 1(b)). We further confrmed the upregula- and S2c). tion of GATA1 in additional 30 pairs of fresh frozen PDAC Colony formation assay confrmed these results (Figures tissues and matched paracancerous tissues with Western blot 2(e) and S2d). To sum up, GATA1 is critical for proliferation analysis (P =0.0011) (Figure 1(c)). Tese fndings suggest that and intrinsic gemcitabine resistance in pancreatic cancer GATA1 expression is highly elevated in PDAC. cells.

3.2. GATA1 Is an Independent Prognostic Factor in PDAC 3.4. Screening for Target Genes Contributing to GATA1- and Predicts Clinical Outcomes of Gemcitabine Terapy. We Mediated Gemcitabine Resistance. To determine the impli- detected the correlation of GATA1 expression with clinical cation of GATA1 in gemcitabine resistance, we established characteristics in a total of 172 PDAC patients. We observed gemcitabine-resistant (Gem-R) CFPAC-1 and PANC-1 cell that patients with highly expressed GATA1 had shorter −5 lines as acquired chemoresistant model through chronic OS (P=0.0001) and RFS (P=8.5×10 )(Figure1(d)).Since gemcitabineexposure(Figure3(a)).WeconfrmedthatGem- gemcitabine is the frst-line drug for PDAC, we determined Rcells(IC50value,CFPAC-1:33.86�M, PANC-1: 146.59 �M) the correlation of GATA1 status with gemcitabine resistance were more resistant to gemcitabine than the parental cells in PDAC. In 172 PDAC patients, 119 of them received (IC50value,CFPAC-1:6.27�M; PANC-1: 38.01 �M) with cell gemcitabine treatment. For these patients, those with highly viability assay (Figures 3(b) and S3a). Next, we analyzed expressed GATA1 showed poorer prognosis for OS (P = the mRNA expression of chemoresistance-related genes with 0.0003) and RFS (P =0.0001) than those with low expressed Gem-R cells. Consistent with previous reports, Bcl-XL, cyclin GATA1. In contrast, the remaining 53 patients who did D2,Bcl-2,Mcl-1,MDR-1,RRM1,andABCG2wereupreg- not receive any chemotherapy demonstrated no signifcant ulated, and dCK was downregulated. Intriguingly, GATA1 diference in their RFS and OS regardless of the GATA1 status expression level was also elevated in Gem-R cells (Figures (Figure 1(e)). Moreover, the univariate and multivariate anal- 3(c) and S3b). Next, we investigated the genes related to yses revealed that grade, node metastasis, vessels invasion, GATA1-mediated gemcitabine resistance. In CFPAC-1 and and GATA1 status were independent prognostic factors of PANC-1 cells, GATA1 overexpression greatly improved Bcl- OS and RFS (Tables S1 and S2). GATA1 expression positively XL level and modestly enhanced cyclin D2, Mcl-1, MDR- associated with grade and vessels invasion, and but it did 1, NF-�b p65, and ABCG2 expression (Figures 3(d) and not associate with sex, age, and other clinical factors (Table S3c). Consistent with mRNA expression level, GATA1 and S3). Taken together, these results indicated the signifcance of Bcl-XL protein level were also enhanced in Gem-R cells GATA1 in prognosis and response to gemcitabine treatment (Figures 3(e) and S3d). Further Western blot assay showed in PDAC. that GATA1 overexpression enhanced Bcl-XL level, and GATA1 knockdown decreased Bcl-XL expression (Figures 3.3. GATA1 Promotes Cell Proliferation and Confers Gem- 3(f) and S3e). Given that GATA1 and Bcl-XL were both citabine Resistance in Pancreatic Cancer Cells. Based on enhanced in Gem-R cells, we performed cell viability assay previous observation, we further determined the efect of the to investigate the efect of GATA1 or Bcl-XL knockdown GATA1 on proliferation and gemcitabine resistance of PDAC on gemcitabine resistance in Gem-R cells. Te IC50 values cells. First of all, we explored the GATA1 status and gemc- of GATA1 knockdown cells (CFPAC-1: 17.55 �M; PANC-1: itabine sensitivity in PDAC cell lines. Compared to GATA1 64.12 �M) and Bcl-XL knockdown cells (CFPAC-1: 7.72 �M; high expression cell lines (SW1990, AsPC-1, and PANC-1), PANC-1: 49.75 �M) were greatly downregulated compared the GATA1 low expression ones (CFPAC-1, BxPC-3, and to control cells (CFPAC-1: 30.88 �M; PANC-1: 153.67 �M), HPDE6c7) are more sensitive to gemcitabine (Figure 2(a)). suggesting GATA1 and Bcl-XL knockdown both sensitized Subsequently, we established stable GATA1 overexpression Gem-R cells to gemcitabine treatment (Figures 3(g) and S3f). and knockdown cell lines. GATA1 overexpression promoted Tese results suggest that GATA1 was involved in gemcitabine cell proliferation in CFPAC-1 and PANC-1. On the contrary, acquired resistance through Bcl-XL in PDAC cells. Journal of Oncology 5

3 3 p=0.0023

2 Normal Cancer 2

1 1 GATA1 scores GATA1

GATA1 scores GATA1

0 0 Normal Cancer Normal Cancer (a) (b)

100 100

5 p=0.0001 p=8.5×10- HR=2.050 HR=2.030 No. 654321 50 50 NCNCNCNCNCNC p GATA1 = 0.0011

-actin 2 Overall survival Recurrence-free survivalRecurrence-free

No. 7 8 9 101112 0 0 1 0 1000 2000 3000 NCNCNCNCNCNC 0 1000 2000 3000

GATA1  -actin GATA1/ Time (Days) Time (Days) -actin 0 GATA1 Low (n=86) GATA1 Low (n=86) GATA1 High (n=86) Normal Cancer GATA1 High (n=86) (c) (d)

100 100

p=0.0003 p=0.0001 HR=2.287 HR=2.335 50 50 Overall survival Recurrence-free survivalRecurrence-free 0 0 0 1000 2000 3000 0 1000 2000 3000 Time (Days) Time (Days) GATA1 Low (n=59) GATA1 Low (n=59) GATA1 High (n=60) GATA1 High (n=60) Patients received gemcitabine-based chemotherapy

100 100

p=0.1671 p=0.2779

50 50 Overall survival Recurrence-free survivalRecurrence-free

0 0 0 1000 2000 3000 0 1000 2000 3000 Time (Days) Time (Days) GATA1 Low (n=27) GATA1 Low (n=27) GATA1 High (n=26) GATA1 High (n=26)

Patients who did not receive chemotherapy (e)

Figure 1: GATA1 is overexpressed in PDAC and confers resistance to gemcitabine in PDAC patients. (a) Representative immunohistochemical staining of GATA1 in 86 pairs of PDAC tissues and adjacent normal pancreas tissues. Scale bar: 50 �m. (b) H scores of GATA1 expression between normal and cancer tissues were compared by Mann-Whitney U test. (c) Representative Western blot assay of tumor tissues and paired adjacent normal tissues. Paired t test was used to compare GATA1 expression between normal tissues and cancer tissues (n = 30). (d) Kaplan-Meier survival curves for overall survival and recurrence-free survival of 172 PDAC patients according to the relative expression of GATA1. (e) Kaplan-Meier survival curves of patients with (n = 119) or without gemcitabine treatment (n = 53).

3.5. GATA1 Regulates Bcl-XL Transcription through Binding on the transcriptional activity of Bcl-XL promoter (from –1171 to Its Promoter in Pancreatic Cancer Cells. Since GATA1 to +50 bp)-luciferase. Knockdown of GATA1 greatly inhibited improved Bcl-XL mRNA and protein level, we investigated the transcriptional activity of Bcl-XL promoter, suggesting whether GATA1 regulates Bcl-XL expression through binding GATA1 bound to Bcl-XL promoter (Figure 4(a)). We then to its promoter. First of all, we detected the efect of GATA1 determined the binding site of GATA1 on Bcl-XL promoter. 6 Journal of Oncology

6c7 120 AsPC-1 CFPAC-1PANC-1 SW1990 BxPC-3HPDE GATA1 100

-actin 80

60 1990 6 AsPC-1 CFPAC-1 PANC-1 SW BxPC-3 HPDE c7 40 Cell Viability (%) Cell Viability 50  44.40±2.49 6.36±0.45 39.45±3.11 70.34±6.16 5.04±0.58 3.62±0.29 (4) ∗ IC ( M) ∗∗ 20 (1)(3) ∗∗ GATA1 (2)(5)(6) /-actin 1.35±0.10 0.82±0.03 1.28±0.03 1.93±0.07 0.78±0.04 0.37±0.04 0 −3 −2.5 −2 −1.5 −1 32.521.510.50−0.5 Gemcitabine concentration (lg M)

AsPC-1(1) SW1990(4) CFPAC-1(2) BxPC-3(5) PANC-1(3) HPDE6c7(6) (a) 1.4 1.0 1.2 (1) (1)(3) 1.0 ∗∗ (1) (2) 0.8 ∗∗ (1) (2) (3) (2) GATA1 (2) GATA1 0.8 0.6 0.6 -actin -actin 0.4 0.4 CFPAC-1 CFPAC-1

Relative cell number Relative 0.2 0.2 0 cell number Relative 01234 0 01234 Days Days Empty Vector(1) 1 GATA1(2) Control shRNA( ) GATA1 shRNA(2) GATA1 shRNA+GATA1-R(3) (b) 200 GATA1 -+ ∗∗

100

Relative colony number (%) number colony Relative 0 CFPAC-1 CFPAC-1 Empty Vector GATA1

200 GATA1 shRNA - + + GATA1-R - - + ∗∗ 100

0 Relative colony number (%) number colony Relative CFPAC-1 CFPAC-1 Control shRNA GATA1 shRNA GATA1 shRNA+GATA1-R (c) Figure 2: Continued. Journal of Oncology 7

120

100 (1) (2) (3) 80 150 60 ∗∗ 40 100 Cell Viability (%) Cell Viability

20 (1)(3) ∗∗ 50 (2) 0 −3 −2.5 −2 −1.5 −1 32.521.510.50−0.5 0 Gemcitabine concentration (lg M) (%) number colony Relative CFPAC-1 CFPAC-1 Gemcitabine concentration (1M) Control shRNA (1) Control shRNA(1) GATA1 shRNA (2) GATA1 shRNA(2) GATA1 shRNA+GATA1-R (3) GATA1 shRNA+GATA1-R(3) (d) (e)

Figure 2: GATA1 promotes cell proliferation and gemcitabine resistance in vitro. (a) Western blot assay of GATA1 in PDAC and normal pancreatic cell lines. Relative expression of GATA1 in PDAC cell lines was quantifed and listed in the table below along with the IC50 value. Te cell viability curves of diferent PDAC cell lines were shown in the right panel. (b) Cell proliferation curves of CFPAC-1 cells stably infected with lentivirus carrying GATA1 (lef panel), GATA1 shRNA, or GATA1 shRNA plus GATA1-R (right panel). Te rescued cell line was established by reexpression of shRNA-resistant GATA1 (GATA1-R) in the GATA1 knockdown cells. GATA1 overexpression and knockdown efects in CFPAC-1 cells were validated by Western blot assay with �-actin as a loading control. (c) Representative images of colony formation assay in stable CFPAC-1 cells as established in (b). Relative colony numbers were quantifed and compared by t test. (d) Cell viability assay of stable CFPAC-1 cells from (b). Cells were treated with a range of concentration of gemcitabine for 48 h before CCK8 test. (e) Representative images of colony formation assay in stable GATA1 knockdown CFPAC-1 cells. Cells were treated with gemcitabine (1 �M) for 48 h. Relative colony numbers were quantifed and compared by t test.

Actually, there are fve putative binding sites of GATA1 on Bcl- assay. GATA1 overexpression upregulated the IC50 value of XL promoter (from –1171 to +50 bp) (Figure 4(b)). Truncated CFPAC-1 (6.3 �Mto17.55�M) and PANC-1 cells (42.431 �M promoter reporters were used to identify the binding site of to 111.74 �M), whereas Bcl-XL knockdown inhibited the IC50 GATA1 on Bcl-XL promoter. Te deletion of site D containing value (CFPAC-1: 0.915 �MandPANC-1:6.858�M) of the GATG motif abolished GATA1-mediated enhancement of cells, and Bcl-XL knockdown abolished the upregulation Bcl-XL promoter transcriptional activity, while deletion of induced by GATA1 overexpression (CFPAC-1: 0.979 �M the other three putative sites did not (Figures 4(b) and S4a). and PANC-1: 8.408 �M).TeIC50valuesindicatedthat Mutated promoter reporter analysis confrmed that site D GATA1 overexpressed cells displayed higher resistance to was responsible for GATA1 modulation of Bcl-XL promoter gemcitabine, and the knockdown of Bcl-XL reversed this reporter activity, since GATA1 increased the activity of the efect (Figures 5(a), 5(b), and S5a). In the PDAC cells with- reporter with the mutated site B or C, but not with the out gemcitabine treatment, GATA1 overexpression inhib- mutated site D in CFPAC-1 and PANC-1 cells (Figures 4(c) ited apoptosis, and knockdown of Bcl-XL efectively abro- and S4b). Moreover, chromatin immunoprecipitation assay gated this efect, suggesting GATA1 inhibited endogenous (ChIP) assay showed that GATA1 was recruited to the region apoptosis by Bcl-XL. Importantly, GATA1 greatly inhibited containing site D, but not the region containing site B and gemcitabine-induced apoptosis of PDAC cells, and Bcl-XL C (Figure 4(d)). In conclusion, these results suggest that knockdown abolished this efect, suggesting GATA1 medi- GATA1 binds to Bcl-XL promoter (-679/-676) to regulate its ated gemcitabine resistance through Bcl-XL (Figures 5(c) transcription. and S5b). Moreover, we detected the expression of apoptotic proteins with Western blot analysis. Decreased expression of 3.6. GATA1 Inhibited Gemcitabine-Induced Apoptosis through Bax, cleaved PARP, cleaved caspase-9, and cleaved caspase-3 Bcl-XL In Vitro. Since GATA1 and Bcl-XL are both involved was observed in GATA1 overexpressed cells, while elevated in gemcitabine resistance of pancreatic cancer cells, and Bcl- expression was observed in Bcl-XL knockdown cells (lanes XL is a target gene of GATA1, we tested whether GATA1 1, 3, and 5). Gemcitabine greatly enhanced the expression of promotes gemcitabine resistance through regulating Bcl- these apoptotic proteins, and GATA1 remarkably ofset this XL level with cell viability assay and colony formation efect (lanes 1, 2, and 4). Downregulation of Bcl-XL abolished 8 Journal of Oncology

120 100 80 60 40

Cell Viability (%) Cell Viability 20 ∗∗ +Gradually increasing Stably passaged in 0 concentration of +Gemcitabine Gemcitabine- −1−2 3210 Gemcitabine containing medium Gemcitabine concentration (lg M) CFPAC-1 WT Gem-R CFPAC-1 Gem-R (a) (b) 6 9 ∗∗ 8 5 ∗∗ 7 4 ∗∗ 6 ∗ 5 3 ∗∗ 4 ∗∗ R 3 ∗∗ ∗ 2 ∗∗ ∗ ∗ ∗ 2 ∗ ∗ ∗ 1 Relative mRNA expression mRNA Relative

Relative mRNA expression mRNA Relative 1 ∗∗ 0 0 CFPAC-1 CFPAC-1 Gem- 2 Bcl-XL 53 53 -XL p cl-1 dCK CG Akt ivin p cl-1 R-1 dCK Akt cl 6 KIP1 c-myc Bcl-2 M 1 CDK6 KIP1 c-myc Bcl-2 M 1 1B CDK MDR-1 RRM hENT1 GATA1Bcl-XLurv MD RRM hENT1 2 GATA Survivin p27 AB S p27 ABCG 1 Cyclin D2 NF-kb p65 Cyclin D2 NF-kb p65 GATA

CFPAC-1 CFPAC-1+EV -actin CFPAC-1 Gem-R CFPAC-1+GATA1 (c) (d) (e)

120 (1) (2) (3) 100 Bcl-XL 80 1 CFPAC-1 GATA 60 -actin 40 ctor Cell Viability (%) Cell Viability (1) siRNA 20 ∗∗ ol (2)(3) r 0 1 Empty Ve GATA1 Cont GATA siRNA −1−2 3210 Bcl-XL Gemcitabine concentration (lg M) CFPAC-1-Gem-R GATA1 Control siRNA(1) 1 -actin GATA siRNA(2) Bcl-XL siRNA(3) (f) (g) Figure 3: Screening for target genes in GATA1 mediated gemcitabine resistance. (a) Schematic representation of the protocol to obtain gemcitabine-resistant PDAC cell line (Gem-R). Red and blue colors of cells represent gemcitabine resistance and sensitivity, respectively. Gem-R cells were stably passaged for more than 10 times afer exposed to an increasing concentration of gemcitabine for over 3 months. (b) Cell viability assays of CFPAC-1 and CFPAC-1 Gem-R cells treated with a range of concentration of gemcitabine for 48 h. (c) qRT-PCR analysis of relative mRNA expression in CFPAC-1 versus CFPAC-1 Gem-R cells of genes related to proliferation and drug resistance. (d) qRT- PCR analysis of relative mRNA expression in CFPAC-1 cells transiently transfected with empty vector or GATA1. Te genes from (c) were used for qRT-PCR. (e) Elevated expression of GATA1 and Bcl-XL was detected in CFPAC-1 Gem-R by Western blot analysis. (f) Western blot analysis of Bcl-XL and GATA1 in CFPAC-1 cells transiently transfected with empty vector or GATA1, control siRNA, or GATA1 siRNA. �-actin was used as a loading control. (g) Cell viability curves of CFPAC-1-Gem-R cells transfected with GATA1 siRNA or Bcl-XL siRNA. Te cells were exposed to a range of concentration of gemcitabine for 48 h before CCK8 assay. Te knockdown efects of siRNAs were confrmed by Western blot analysis, with �-actin as a loading control. All data shown are means ± SD of three independent experiments with triplicate ∗ ∗∗ each, � <0.05; � <0.01. the efect of GATA1 on these apoptotic proteins no matter completely abolished GATA1 conferred gemcitabine resist- with or without gemcitabine treatment (lane 5-8) (Figures ance (Figure 6(a)). IHC assay verifed the overexpression 5(d) and S5c). In conclusion, GATA1 decreased endogenous of GATA1 and knockdown of Bcl-XL in xenograf tumors and gemcitabine induced apoptosis through Bcl-XL. (Figure 6(b)). TUNEL assay was used to determine the apoptosis index of xenograf tumors. GATA1 overexpression 3.7. GATA1 Mediates Gemcitabine Resistance of PDAC through decreased the proportion of TUNEL-positive cells, and Bcl-XL In Vivo. We further confrmed GATA1 mediated Bcl-XL knockdown increased the ratio of TUNEL-positive gemcitabine resistance with xenograf mouse model. GATA1 cells. Moreover, the efect of GATA1 on apoptotic index overexpression markedly promoted tumor growth, whereas was abrogated by Bcl-XL knockdown. Gemcitabine greatly knockdown of Bcl-XL dramatically inhibited pancreatic enhanced the proportion of TUNEL-positive cells, and tumor growth, and Bcl-XL knockdown ofset GATA1 medi- GATA1 overexpression decreased gemcitabine-induced ated tumor growth. Upon the treatment of gemcitabine, apoptosis signifcantly, whereas knockdown of Bcl-XL we observed that GATA1 greatly counteracted gemcitabine- abrogated the antiapoptotic efect of GATA1 (Figure 6(c)). induced tumor shrink. However, Bcl-XL knockdown Te results need to be confrmed with orthotopic Journal of Oncology 9

1.2 1.0 (1) (2) (3) 0.8 GATA1 0.6 ∗ -actin 0.4 ∗∗ 0.2 0 Bcl-XL Promoter +1 Relative Luciferase Activity Luciferase Relative CFPAC-1 -1171 AATC GATC GATC GATG GATC +50 Control siRNA(1) GATA1 siRNA1(2) -37 GATA1 siRNA2(3) -1117 -954 -936 -679

Relative Luciferase Activity 10 12 14 16 18

1.2 +1 0 2 4 6 8 (1) (2) (3) 1.0 -1171 Empty Vector

Luc ∗∗ 0.8 GATA1 -1029

0.6 Luc ∗∗ -actin 0.4 ∗∗ -710 ∗∗ 0.2 Luc ∗∗

0 -438 GATA1

Relative Luciferase Activity Luciferase Relative PANC-1 Luc

Control siRNA(1) Luc GATA1 siRNA1(2) GATA1 siRNA2(3) CFPAC-1 (a) (b)

Bcl-XL Promoter +1 -1171 GATC GATC GATG +50

-954 -936 -679   Putative GATA1-binding site B: 5 -...CCAGATCTAC...-3   Mutated GATA1-binding site B: 5 -...CCATCAATAC...-3   Putative GATA1-binding site C: 5 -...GGCGATCTCT...-3   Mutated GATA1-binding site C: 5 -...GGCTCAGTCT...-3   Putative GATA1-binding site D: 5 -...GCCGATGAAG...-3   Mutated GATA1-binding site D: 5 -...GCCTCGTAAG...-3 12

Relative Luciferase 10 Activity12 14 0 2 4 6 8 +1 10 ∗∗ Empty Vector Luciferase ∗∗ 8

Luciferase ∗∗ 6 4

Luciferase ∗∗ 2 GATA1 Relative Enrichment Relative Luciferase 0 -actin Site B Site C Site D Luciferase CFPAC-1 IgG CFPAC-1 GATA1 (c) (d)

Figure 4: GATA1 regulates Bcl-XL transcription through binding to its promoter. (a) Luciferase reporter assay of CFPAC-1 and PANC-1 cells cotransfected with the Bcl-XL promoter-Luc reporter and control siRNA or GATA1 siRNAs. Representative Western blot analysis indicates the expression of GATA1. (b) Relative luciferase activity of diferent truncated Bcl-XL promoter reporter constructs in CFPAC-1 cells transfected with empty vector or GATA1. A, B, C, D, and E indicate putative binding sites of GATA1. (c) Relative luciferase activity of wild-type and mutated Bcl-XL promoter reporter constructs in CFPAC-1 cells transfected with empty vector or GATA1. B, C, and D indicate putative binding sites of GATA1. Te “X” symbol denotes mutated GATA1-binding sites. (d) ChIP analysis of the occupancy of GATA1 protein on ∗ ∗∗ putative GATA1-binding sites. All values shown are means ± SD of three independent experiments with triplicate each, � <0.05; � <0.01 versus corresponding control. 10 Journal of Oncology

120 120 100 (1) (2) (3) (4) (1) (2) (3) (4) 100 Bcl-XL Bcl-XL 80 80 GATA1 GATA1 60 60 -actin -actin 40 40 (2) Cell Viability (%) Cell Viability

Cell Viability (%) Cell Viability ∗∗ (2) 20 ∗∗ (1) (1) ∗∗ 20 ∗∗ (3)(4) (3)(4) 0 0 −1−2−3 3210 −1−2−3 3210 Gemcitabine concentration (lg M) Gemcitabine concentration (lg M) CFPAC-1 PANC-1 EV+Control shRNA(1) EV+Control shRNA(1) GATA1+Control shRNA(2) GATA1+Control shRNA(2) EV+Bcl-XL shRNA(3) EV+Bcl-XL shRNA(3) GATA1+Bcl-XL shRNA(4) GATA1+Bcl-XL shRNA(4) (a)

GATA1 - + - + Bcl-XL shRNA - - + +

200 ∗∗ ∗∗ Control ∗∗

100 ∗∗ ∗∗ ∗∗ Gemcitabine

Relative colony number (%) number colony Relative 0 CFPAC-1 Control Gemcitabine EV+Control shRNA GATA1+Control shRNA EV+Bcl-XL shRNA GATA1+Bcl-XL shRNA (b)

GATA1 --+ + Bcl-XL shRNA --+ + 7 7 7 7 Q2 60 10 Q2 Q2 10 10 Q1 10 Q1 Q1 Q1 Q2 ∗∗

6 6 13.1 0.61 6 9.39 1.99 3.18 6 0.11 0.90 2.77 ∗∗ 10 10 10 10 50 ∗∗ 5 5 5 5 10 10 10 Control 10 4 4 4 4 40 10 10 10 10 3 3 3 3 Q4 Q4 10 10 10 Q3 Q4 Q3 Q4 Q3 10 Q3 30 2 95.7 2 81.8 4.24 2 81.5 6.36

2 90.4 4.47 3.63

10 10 10 ∗∗ 10 2 3 4 5 6 7 2 3 4 5 6 7 2 3 4 5 6 7 2 3 4 5 6 7

PI 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ∗∗

7 20 7 7 7 ∗ 10 10 10 10 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Level Apoptosis 6 6 4.21 20.9 6 0.15 9.12 6 6.92 26.7 6.43 27.1 10 10 10 10 10 5 5 5 5 10 Gemcitabine 10 10 10

4 0 4 4 4 10 10 10 10 Control Gemcitabine 3 3 Q3 3 Q3 3

Q4 Q4 Q4 10 Q4

10 Q3 Q3 9.04 10 9.93 10 65.8 80.8 47.2 2 51.0 15.5 2 2 2 19.2

10 EV+Control shRNA 10 10 10 2 3 4 5 6 7 102 103 104 105 106 107 102 103 104 105 106 107 102 103 104 105 106 107 10 10 10 10 10 10 GATA1+Control shRNA Annexin V-APC EV+Bcl-XL shRNA CFPAC-1 GATA1+Bcl-XL shRNA (c)

Figure 5: Continued. Journal of Oncology 11

1 GATA1 - - + + - - + + GATA - - + + - - + +

BCL-XL shRNA - - - - + + + + BCL-XL shRNA - - - - + + + + Gem - + - + - + - + Gem - + - + - + - +

cleaved Caspase3 cleaved Caspase3

cleaved Caspase9 cleaved Caspase9

Cleaved PARP Cleaved PARP

Bax Bax

Bcl-XL Bcl-XL

GATA1 GATA1

-actin -actin CFPAC-1 PANC-1 (d)

Figure 5: GATA1 inhibits gemcitabine-induced apoptosis through Bcl-XL in vitro. (a) Cell viability curves of CFPAC-1 and PANC-1 cells stably infected with the indicated lentiviruses. Western blot assay of GATA1 and Bcl-XL expression in indicated cells. �-actin was used as a loading control. Cells were treated with a range of concentration of gemcitabine for 48 h before CCK8 assay. (b) Representative images of colony formation assays in stable CFPAC-1 cells lines from (a). Te cells were treated with control or gemcitabine (1 �M) for 48 h before seeded into 6-well plates. Relative colony numbers were quantifed and compared by t test. (c) Representative images of fow cytometry analysis of apoptosis in CFPAC-1 cells transfected with indicated lentivirus. Te cells were treated with or without gemcitabine (5 �m) for 48 h. Statistical analysis of apoptosis rates was shown in the right panel. (d) Western blot analysis of GATA1, Bcl-XL, Bax, cleaved PARP, cleaved caspase 9, and cleaved caspase 3 in CFPAC-1 and PANC-1 infected with the indicated lentivirus. Te cells were treated with or without gemcitabine (10 �M for CFPAC-1 and 50 �M for PANC-1) for 48 h before analysis. All data shown are means ± SD of three independent experiments with ∗ ∗∗ triplicate each, � <0.05; � <0.01.

nude mouse model. Taken together, GATA1 mediates 4. Discussion gemcitabine resistance through Bcl-XL related antiapoptosis in vivo. Gemcitabine remains a cornerstone for PDAC treatment since1997;however,theintrinsicandacquiredresistanceto this chemotherapy inhibits its function and leads to poor 3.8. Bcl-XL Positively Correlates with GATA1 and Is a Prognos- outcome of patients [28]. Although multiple mechanisms tic Marker for Gemcitabine Treatment in PDAC. As GATA1 of gemcitabine resistance have been reported, no signifcant binds to Bcl-XL promoter and regulates Bcl-XL expression, advances have been achieved to improve the prognosis of we subsequently determined the correlation of GATA1 and PDAC patients. Terefore, a novel target to enhance current Bcl-XL in PDAC samples. We verifed the specifcity of the chemotherapy is clearly needed to improve the outcomes of Bcl-XL antibody used in IHC by antigen competition and patients with pancreatic cancer. lysates of Bcl-XL knockdown cells (Figures S6a and S6b). In the current study, we identifed a novel role of GATA1 Te IHC assay indicated that expression of GATA1 positively in tumor proliferation and gemcitabine resistance in PDAC. correlated with Bcl-XL expression (Figures 7(a) and 7(b)). Firstly, we found that, compared to normal pancreatic tis- PDAC patients with high Bcl-XL expression had shorter sue, GATA1 was overexpressed in PDAC tissues, and the OS (P=0.0029) and RFS (P=0.0004) (Figure 7(c)). Next, we expression of GATA1 was correlated with overall survival, detected the efect of Bcl-XL expression on gemcitabine recurrence-free survival, and gemcitabine response. Sec- response in PDAC. For patients who received gemcitabine- ondly, GATA1 upregulated Bcl-XL transcription by binding to based chemotherapy, those with highly expressed Bcl-XL its promoter. Tirdly, GATA1 induced intrinsic and acquired showed poorer prognosis for OS (P = 0.0008) and RFS (P resistance to gemcitabine in PDAC cells through enhancing =0.0012) than those with low Bcl-XL expression. In contrast, Bcl-XL mediated antiapoptosis. Moreover, Bcl-XL expression patients who did not receive any chemotherapy demonstrated was positively correlated with GATA1 and predicted clinical no signifcant diference in their RFS and OS between high outcomes and gemcitabine response in PDAC patients. Tese and low Bcl-XL expression (Figure 7(d)). Moreover, Bcl-XL fndings indicated that GATA1 and Bcl-XL may be potential status was proved to be an independent prognostic factor therapeutic targets to improve prognosis of PDAC patients. according to the univariate and multivariate analyses (Tables Previous reports about GATA1 mainly focused on its S1 and S2). In conclusion, these data indicated that Bcl-XL function in erythroid and megakaryocytic cells. GATA1 has a major role in the resistance of gemcitabine treatment in regulates the diferentiation, proliferation, and apoptosis of PDAC. erythroid and megakaryocytic cells [23]. Mutation of GATA1 12 Journal of Oncology

(1)

2500 (2)

2000 (3) ) 3 (2) (4) 1500 ∗

(6) (5) 1000 (1) ∗ ∗ (6) (4) 500 (5) ∗∗ Tumor Volume (mm (3) ∗ (7) (8) ∗ 0 (7) 0 3 6 9 121518212427 (8) Days post-treatment initiation Gem- EV+Control shRNA (1) Gem- GATA1+Control shRNA (2) Gem- EV+Bcl-XL shRNA (3) Gem- GATAI+Bcl-XL shRNA (4) Gem+ EV+Control shRNA (5) Gem+ GATA1+Control shRNA(6) Gem+ EV+Bcl-XL shRNA (7) Gem+ GATA1+Bcl-XL shRNA (8) (a)

Gem- EV+Control shRNA (1) Gem- GATA1+Control shRNA (2) Gem- EV+Bcl-XL shRNA (3) Gem- GATAI+Bcl-XL shRNA (4) Gem+ EV+Control shRNA (5) Gem+ GATA1+Control shRNA(6) Gem+ EV+Bcl-XL shRNA (7) Gem+ GATA1+Bcl-XL shRNA (8)

GATA1

Bcl-XL

(b) (1) (2) (3) (4) (5) (6) (7) (8)

TUNEL

50 ∗∗ 40 ∗∗ DAPI ∗∗ 30

20 ∗∗ ∗∗ Merge 10 ∗ Apoptosis index (%) Apoptosis 0 1 234 5678 (c)

Figure 6: GATA1 confers gemcitabine resistance of PDAC through Bcl-XL in vivo. (a) Volume of xenografs tumors derived from PANC-1 cells infected with the indicated lentivirus and treated with gemcitabine or control. Te tumors were measured by vernier caliper every 3 days and the tumor growth curves were plotted. Tumor volumes were presented as means ± SD (n = 6). Images of all xenografs tumors excised at day ∗ ∗∗ 27 afer treatment were shown in the right. � <0.05; � <0.01. (b) Representative immunohistochemical staining of GATA1 and Bcl-XL in xenografs tumors of the indicated groups from (a). Scale bar: 50 �m. (c) Representative images of apoptotic cells visualized by tunnel staining and counterstained by DAPI in tumor sections of the indicated groups from (a). Scale bar: 50 �m. Apoptosis index was quantifed ∗ ∗∗ and analyzed. Data are shown in means ± SD. � <0.05; � <0.01.

leads to acute megakaryoblastic leukemia (AMKL) in infants normal tissues [24, 30]. Furthermore, GATA1 enhanced with Down syndrome and transient myeloproliferative dis- breast cancer cells invasion and angiogenesis through pro- order (TMD). Knockdown of GATA1 to 5% of its wild-type moting epithelial-mesenchymal transition (EMT) [31] and expression level causes high incidence of erythroid leukemia VEGF expression [26]. Here, we found a novel function of in mice [29]. Recently, more and more researches indicated GATA1 in gemcitabine resistance in pancreatic cancer. High that GATA1 played an important role in solid cancer. In expression of GATA1 is associated with poor prognosis of breast cancer and colorectal cancer, GATA1 was reported PDAC patients treated with gemcitabine, indicating GATA1 to be overexpressed compared with matched adjacent was correlated with gemcitabine resistance in PDAC patients. Journal of Oncology 13

r=0.4179 8 P=1.162×10- 3

Case1 Case2

2 GATA1 Scores Bcl-XL 1

Bcl-XL 0 3210 GATA1 Scores (a) (b)

100 100

p=0.0029 p=0.0004 HR=1.881 HR=1.903

50 50 Overall survival Recurrence-free survivalRecurrence-free

0 0 0 1000 2000 3000 0 1000 2000 3000 Time (Days) Time (Days)

Bcl-XL High (n=86) Bcl-XL High (n=86) Bcl-XL Low (n=86) Bcl-XL Low (n=86) (c)

100 100

p=0.0008 p=0.0012 HR=2.111 HR=2.030

50 50 Overall survival Recurrence-free survivalRecurrence-free

0 0 0 1000 2000 3000 0 1000 2000 3000 Time (Days) Time (Days)

Bcl-XL High (n=62) Bcl-XL High (n=62) Bcl-XL Low (n=57) Bcl-XL Low (n=57)

Patients received gemcitabine-based chemotherapy 100 100

p=0.3228 p=0.1775

50 50 Overall survival Recurrence-free survivalRecurrence-free

0 0 0 1000 2000 3000 0 1000 2000 3000 Time (Days) Time (Days)

Bcl-XL High (n=24) Bcl-XL High (n=24) Bcl-XL Low (n=29) Bcl-XL Low (n=29)

Patients who did not receive chemotherapy (d) Figure 7: Continued. 14 Journal of Oncology

Gemcitabine Human nucleoside transporters

NH2

CARD N Apaf1 Casp 9 PTP O N Gemcitabine HOCH2 O Bak F OH F Apaf1 Apaf1 CARD Casp 9 Casp 9 CARD Bax Casp 9 dFdCMP

CARD Bax

Casp 3 Apaf1 dFdCDP Bcl-XL Mitochondrial Other pathway Parp Substrates apoptosis dFdCTP

Apoptosis

GATA1 GATG Bcl-XL DNA damage

DNA synthesis Bcl-XL promoter region

(e) Figure 7: Bcl-XL positively correlates with GATA1 and is a prognostic marker for PDAC. (a) Representative immunohistochemical staining of GATA1 and Bcl-XL in human PDAC tissues. Scale bar: 50 �m. (b) Correlation between GATA1 and Bcl-XL expression was analyzed in 172 PDAC samples (from Figure 1(d)) by Spearman rank correlation analysis. (c) Kaplan-Meier survival curves for overall survival and recurrence-free survival of 172 PDAC patients (from Figure 1(d)) according to the relative expression of Bcl-XL. (d) Kaplan-Meier survival curves of patients with (n=119) or without gemcitabine treatment (n = 53). (e) Graphic summary of GATA1 promoting gemcitabine resistance through antiapoptotic pathway. Gemcitabine causes apoptosis in mitochondrial pathway owing to DNA synthesis inhibition followed by DNA damage. GATA1 acts as a transcription activator by binding to the “GATG” site of Bcl-XL promoter region, leading to upregulation of Bcl-XL expression. Elevated Bcl-XL expression can counteract gemcitabine-induced apoptosis via binding to Bax, preventing the release of cytochrome c and thus preventing caspase activation.

Te PDAC cell lines (SW1990, AsPC-1, and PANC-1) that resistance through activating Bcl-XL. GATA1 enhanced Bcl- displayed relatively higher basal levels of GATA1 are also XL expression through binding to its promoter and then more resistant to gemcitabine. Based on these data, we promoted Bcl-XL mediated antiapoptosis, leading to gem- may assume that GATA1 is correlated with intrinsic gemcitabine resistance in PDAC cells (Figure 7(e)). Te levels citabine resistance. On the other hand, GATA1 expression of GATA1 and Bcl-XL were both upregulated in established was upregulated in established gemcitabine-resistant cell gemcitabine-resistant cells. Moreover, Bcl-XL knockdown lines, suggesting GATA1 was also associated with acquired sensitized the Gem-R cells to gemcitabine treatment, sug- chemoresistance. gesting Bcl-XL was involved in GATA1-induced acquired Te activation of antiapoptotic genes might be respon- gemcitabine resistance. Also, Bcl-XL knockdown greatly sible for acquired resistance of pancreatic cancer cells to sensitized the tumors to gemcitabine in vivo.However,the gemcitabine. Mutated p53 confers resistance to gemcitabine subcutaneous xenograf models could not fully mimic the as the mutation abolished its function in inhibiting Bcl- physiological tumor development. Te efect of Bcl-XL on XL expression [32]. Downregulation of BNIP3, which could gemcitabine resistance needs further confrmation with the antagonize the activity of antiapoptotic proteins such as orthotopic models. Te high expression of both GATA1 and Bcl-2 and Bcl-XL and promote apoptosis, associated with Bcl-XL correlates to poor clinical outcomes in patients treated intrinsic chemoresistance to gemcitabine in PDAC cells [33]. with gemcitabine, suggesting GATA1 and Bcl-XL could In this research, we elucidated a novel pathway of gemcitabine be used as prognostic markers for gemcitabine treatment. Journal of Oncology 15

Furthermore, GATA1 inhibition alone or in combination with S6: validation of Bcl-XL antibody specifcity; Table S1: Cox Bcl-XL inhibition of may be a useful strategy for treating univariate and multivariate analysis of overall survival in gemcitabine-resistant PDAC patients with high GATA1 level. PDAC patients; Table S2: Cox univariate and multivariate In addition to conferring gemcitabine resistance, GATA1 analysis of recurrence-free survival in PDAC patients; Table was also showed to promote cancer cell proliferation in vitro S3: the relationship of GATA1 and Bcl-XL with clinical char- and in vivo. Mechanistically, cyclin D2, frequently thought to acteristics in PDAC patients; Table S4: sequences for shRNA, play a critical role in promoting tumor cell proliferation [34], qRT-PCR, and ChIP primers. (Supplementary Materials) was upregulated in GATA1 overexpressed cells. We speculate that GATA1 confers gemcitabine resistance in pancreatic References cancer not only by activating Bcl-XL, but also by retaining proliferative potential of PDAC cancer following treatment [1] M. Hidalgo, “Pancreatic cancer,” Te New England Journal of with gemcitabine. In conclusion, cyclin D2 may play a Medicine,vol.362,no.17,pp.1605–1617,2010. substantial role in GATA1 mediated gemcitabine resistance. [2]R.L.Siegel,K.D.Miller,andA.Jemal,“Cancerstatistics,2017,” In summary, we characterized the signifcance of GATA1/ CA: A Cancer Journal for Clinicians,vol.67,no.1,pp.7–30,2017. Bcl-XL axis in gemcitabine resistance in PDAC cells, and [3] P. Michl and T. M. Gress, “Current concepts and novel targets inhibition of this axis in PDAC cells enhanced the sensitivity in advanced pancreatic cancer,” Gut,vol.62,no.2,pp.317–326, of gemcitabine. Importantly, GATA1 and Bcl-XL both predict 2013. clinical outcomes of gemcitabine therapy. Tese fndings [4] H. Oettle, “Progress in the knowledge and treatment of might provide a promising strategy and target to develop new advanced pancreatic cancer: From benchside to bedside,” Can- therapy against pancreatic cancer. cer Treatment Reviews,vol.40,no.9,pp.1039–1047,2014. [5]S.Dhayat,W.A.Mardin,S.T.Mees,andJ.Haier,“Epigenetic 5. Conclusions markers for chemosensitivity and chemoresistance in pancreatic cancer-a review,” International Journal of Cancer,vol.129, Taken together, we demonstrated that GATA1 is a new no. 5, pp. 1031–1041, 2011. predictive marker for prognosis and gemcitabine resistance in [6] M. Amrutkar and I. P. Gladhaug, “Pancreatic cancer chemore- pancreatic cancer patients. We found that GATA1 is involved sistance to gemcitabine,” Cancers,vol.9,no.11,article157,2017. in both intrinsic and acquired gemcitabine resistance in [7]K.Giehl,B.Skripczynski,A.Mansard,A.Menke,andP. 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