Cancer Letters 373 (2016) 130–137

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

MicroRNA-101-3p reverses gemcitabine resistance by inhibition of ribonucleotide reductase M1 in pancreatic cancer Pei Fan a,b,c, Li Liu a,b,c, Yefeng Yin a,b,c, Zhefu Zhao a,b,c, Yiyao Zhang a,b,c, Prince S. Amponsah a,b,c, Xi Xiao a,b,c, Nathalie Bauer a,b,c, Alia Abukiwan a,b,c, Clifford C. Nwaeburu a,b,c, Jury Gladkich a,b,c, Chao Gao b,c, Peter Schemmer b,c, Wolfgang Gross a,b,c, Ingrid Herr a,b,c,* a

Molecular OncoSurgery, University of Heidelberg, Heidelberg, Germany Section Surgical Research, University of Heidelberg, Heidelberg, Germany c Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany b

A R T I C L E

I N F O

Article history: Received 6 December 2015 Received in revised form 20 January 2016 Accepted 22 January 2016 Keywords: Epigenetics microRNA Chemoresistance

A B S T R A C T

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal malignancies and resistance to chemotherapy prevents the therapeutic outcome. MicroRNAs provide a novel therapeutic strategy. Here, the established and primary human PDA cell lines PANC-1, AsPC-1, MIA-PaCa2, AsanPaCa, BxPC-3 and three gemcitabine-resistant subclones were examined. A gene expression profiling revealed that the ribonucleotide reductase M1 (RRM1) was upregulated in gemcitabine-resistant cells, which was confirmed by qRT-PCR, Western blot analysis and immunostaining. Inhibition of RRM1 by lipotransfection of siRNA reduced its expression and reversed gemcitabine resistance. The expression of RRM1 correlated to gemcitabine resistance in vitro and was higher in malignant patient pancreas tissue compared to nonmalignant pancreas tissue. By microRNA expression profiling, we identified microRNA-101-3p as topdownregulated candidate. Lipotransfection of microRNA-101-3p mimics inhibited the expression of RRM1, reduced the luciferase activity of its 3’UTR and sensitized for gemcitabine-induced cytotoxicity. These results underline the relevance of microRNA-101-3p-driven regulation of RRM1 in drug resistance and suggest the co-delivery of microRNA-101-3p and gemcitabine for more effective therapy outcome. © 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction Pancreatic cancer is the 7th most common cause of cancer death worldwide and pancreatic ductal adenocarcinoma (PDA) accounts for 90% of all pancreatic cancers [1]. Due to the low rate of early detection, most of patients lose the option for curative surgery. Gemcitabine is considered as the standard chemotherapy [2] in the past decades. Chemotherapy resistance, either intrinsic or acquired, is a key clinical problem of treatment failure in PDA [3]. Several pathways involved in apoptosis, drug efflux, growth, differentiation, proliferation and angiogenesis appear to influence chemosensitivity [4]. However, the overcoming of chemoresistance in patients is still a challenge, and the disclosure of the underlying mechanisms is essential to solve this problem.

Abbreviations: MiR, microRNA; PDA, pancreatic ductal adenocarcinoma; 3’UTR, 3′-untranslated region. * Corresponding author. Tel.: +49 6221 56 6401; fax: +49 6221 56 6402. E-mail address: [email protected] (I. Herr). http://dx.doi.org/10.1016/j.canlet.2016.01.038 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

Recently, epigenetic approaches demonstrated an important role of microRNAs (miRs) in chemoresistance [5]. MiRs are usually 18– 25 nucleotides in length and participate in transcriptional and posttranscriptional regulation of gene expression by base pairing to the 3′-untranslated region (3’UTR) of target genes. For instance, the overexpression of miR-1246 increased the tumor-initiating potential, induced drug resistance and predicted a worse prognosis in pancreatic cancer by inhibition of CCNG2 expression [6]. Upregulation of miR-200 reversed epithelial-to-mesenchymal transition in chemoresistant pancreatic cancer cells by down-regulation of ZEB1, slug, and vimentin [7]. However, the role of miRs in sensitivity of tumor cells to chemotherapy is not fully understood and needs to be further investigated. Ribonucleotide reductase M1 (RRM1), encodes one of two subunits of ribonucleotide reductase, an enzyme, which is essential for DNA synthesis [8]. This enzyme converts ribonucleoside diphosphates to deoxyribonucleoside diphosphates [9]. Gemcitabine inhibits DNA elongation processes by incorporating into DNA as faulty dCDP, which is due to the chemical nature of gemcitabine, namely 2′,2′difluoro deoxycytidine [10,11]. Some resistant tumor cells, including non-small-cell lung cancer, intrahepatic carcinoma, gastric cancer

P. Fan et al./Cancer Letters 373 (2016) 130–137

and pancreatic cancer [12–18], demonstrated enhanced levels of RRM1, which resulted in a higher dCTP synthesis [8], to compete with gemcitabine. In the present study, we identified the inhibition of miR-1013p in gemcitabine-resistant PDA cells as key regulator of RRM1 upregulation and detected two miR-101-3p binding sites in the RRM1 3’UTR. Gemcitabine-resistant cells had low levels of miR-101-3p and the lipofection of miR-101-3p mimics led to inhibition of RRM1 expression and restored gemcitabine sensitivity. Materials and methods Cell culture The established human PDA cell lines AsPC-1, PANC-1, MIA-PaCa2 and BxPC-3 were obtained from the American Type Culture Collection (ATCC) and the primary cell line ASAN-PaCa from the European Pancreas Center (EPC). Gemcitabineresistant BxPC-3 cells (Bx-GEM) were selected by continuous treatment of BxPC-3 cells in increasing concentrations of gemcitabine when the confluence of cells reached 40~60% resulting in subclones resistant to 50 nM (Bx-GEM50), 100 nM (BxGEM100) or 200 nM gemcitabine (Bx-GEM200) as described [19]. All cell lines were cultured in DMEM (18 mmol/L glucose) supplemented with 10% FCS and 5% HEPES. The established cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany). Mycoplasma-negative cultures were ensured by monthly mycoplasma tests. Patient tissues Patient tissues were obtained under the approval of the ethical committee of the University of Heidelberg after receiving written informed consent from the patients. The diagnoses were established by conventional clinical and histological criteria according to the World Health Organization (WHO). All surgical resections were indicated by the principles and practice of oncological therapy. Reagents Gemcitabine solution (Eli Lilly, Indianapolis, IN, USA) was diluted in cell culture medium to a 100-μM stock and then added directly to cell culture medium according to the purposes of the experiments. The final concentrations of the solvents in medium were 0.1% or less. Cell viability Viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2, diphenyltetrazolium bromide (MTT) as described [20].

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Abcam, Cambridge, UK) and mouse monoclonal to β-Actin (Sigma-Aldrich, St. Louis, MO, USA). Immunofluorescence and immunohistochemistry The staining of in vitro cultured cells or of paraffin-embedded patient tissue was performed as described [21]. Lipotransfection MiR mimics or siRs were transfected to cells with HiPerFEct Transfection Reagent (Qiagen, Hilden, Germany) according to instructions of the manufacturer. For Mocktransfection, the AllStars Negative Control siRNA was used. The miR and siRs were from Quiagen (Hilden, Germany): siRN RRM1: Sense: 5′-GCAUGUCUUCAGUAGCCAATT3′; Antisense: 5′-UUGGCUACUGAAGAC-AUGCTG-3′. miR-101-3p mimic: 5′-UACA GUACUGUGAUAACUGAA-3′. Dual-luciferase reporter assay A pLightSwitch_3’UTR luciferase vector carrying the full length of the human RRM1 3′-untranslated region (pLightSwitch_RRM1_3UTR_Renilla, 656 bp) was from Bio-Cat (Heidelberg, Germany). Cells were seeded into 96-well microplates (Thermo Fisher Scientific GmbH, Dreieich, Germany) at 2 × 104 cells/well and transfected with a mixture of 50 ng pLightSwitch_RRM1_3UTR_renilla vector, 25 ng firefly luciferase in pSGG vector (Promega, Mannheim, Germany) and 5 pmol mimic or Allstar negative control miR mimic using lipofectamine 2000 (Thermo Fisher Scientific GmbH, Dreieich, Germany). Both Renilla and firefly luciferase activities were measured 24 hours after transfection with the Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) using a BMG FLUOstar OPTIMA Microplate Reader (BMG LABTECH GmbH, Ortenberg, Germany). The relative Renilla luciferase activities were analyzed according to the instructions of the manufacturer (Promega, Mannheim, Germany). Statistical evaluation Data obtained with established cell lines are presented as the means ± SD from at least three separate experiments, each performed in triplicate or octuplicate (MTT). The significance of the data was analyzed using the Student’s t-test for parametric data and the Mann–Whitney test with Bonferroni corrections for nonparametric data. P < 0.05 was considered statistically significant (**P < 0.01, *P < 0.05).

Results RRM1 mediates gemcitabine resistance

5-

Apoptosis Cells were stained with FITC-conjugated Annexin V (BD Biosciences, Heidelberg, Germany) and propidium iodide (5 mg/ml) and analyzed by Flow Cytometer (guava easyCyte™ Single Sample Flow Cytometer, Millipore, Darmstadt, Germany), as described [21]. mRNA and miR expression profiling The RNeasy Mini Kit or the miRNeasy Mini Kit was used according to the instructions of the manufacturer (Quiagen, Hilden, Germany) and the expression profiling was performed at the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ) Heidelberg, using the Human HT-12 v4 Expression Bead Chip Kit or the Human miR Microarray (Release 19.0). qRT-PCR The RNA concentrations were measured with a NanoDrop 2000 Spectrophotometer (Nano Drop Technologies, Wilmington, USA) and 500 ng total RNA or miRNA were reverse transcribed to cDNA using the High Capacity RNA to cDNA KIT (Thermo Fisher Scientific GmbH, Dreieich, Germany) or the Taqman microRNA Reverse Transcription kit (Thermo Fisher Scientific GmbH, Dreieich, Germany). Real time PCR was performed using the Taqman Gene Expression master mix (Thermo Fisher Scientific GmbH, Dreieich, Germany) or the Taqman Universal PCR master mix (Thermo Fisher Scientific GmbH, Dreieich, Germany). Primers for miR-101-3p, RNU6B, RRM1 and GAPDH were from Thermo Fisher Scientific GmbH (Dreieich, Germany). Western blot analysis Whole-cell protein extracts were prepared and detected by Western blot analysis as described [21]. Antibodies used were rabbit polyclonal to RRM1 (ab81085

To determine the degree of resistance of the BxPC-3-derived gemcitabine-resistant subclone Bx-GEM200, the parental and daughter cells were treated with different concentrations of gemcitabine. The viability and apoptosis were determined 72 h later by MTT assay and staining with annexin-V-FITC/propidium iodide followed by FACS analysis. Whereas gemcitabine totally inhibited the viability of parental BxPC-3 cells, Bx-GEM200 cells were totally resistant, even with the highest gemcitabine concentration of 200 nM (Fig. 1A). Correspondingly, the percentage of apoptosis increased strongly in BxPC-3 cells after gemcitabine treatment, whereas Bx-GEM200 cells were totally resistant (Fig. 1B). To detect differences in gene expression between BxPC-3 and Bx-GEM200 cells, an mRNA expression profiling was performed. Of all 44,052 genes, 9235 mRNAs showed significant differential expression (P < 0.01) including 4873 mRNAs downregulated and 4362 mRNAs upregulated and the most significantly top 12 up- and top 12 downregulated mRNAs are shown (Table 1). To identify genes related to gemcitabine resistance, a literature search was performed in PubMed and Web of Science with the key words “pancreatic cancer”, “gemcitabine” and “chemoresistance” (Table S1). Then, the top 12 down- and the top 12 upregulated genes were combined with the genes identified by literature search (Table S1) and a heat map was created. By this way, RRM1 was identified as the top upregulated gene in Bx-GEM200 cells (Fig. 2A). This result was confirmed qRT-PCR, Western blot analysis and immunofluorescence staining (Fig. 2B). To further elucidate these results, specific siRNA oligonucleotides for inhibition of RRM1 (siRRM1) were lipofected in Bx-GEM200 cells. AllStars nonsense siR was used as control. Seventy-two hours after transfection, the ex-

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PCR (Fig. 3A) and Western blot analysis (Fig. 3B). Whereas ASANPaCa, BxPC-3, PANC-1 and AsPC-1 cells were sensitive to gemcitabine, resistance was increased in MIA-PaCa2 and highest in Bx-GEM200 cells. These findings match with the RRM1 mRNA (Fig. 3C) and protein expression (Fig. 3D), which was low in ASAN-PaCa, BxPC3, PANC-1 and AsPC-1 cells, but increased in MIA-PaCa2 and highest in Bx-GEM200 cells. To further highlight this conclusion, we examined the expression of RRM1 in three different Bx-GEM subclones with a differential grade of resistance to 50 (Bx-GEM50), 100 (BxGEM100) or 200 nM (Bx-GEM20) gemcitabine (Fig. S1A,B, Fig. 3B). The level of RRM1 mRNA and protein expression directly correlated to the level of gemcitabine resistance, suggesting that RRM1 expression directly correlates to the grade of gemcitabine resistance. Fig. 1. Long-time gemcitabine treatment induces resistance. (A) BxPC-3 cells and the derived long-time gemcitabine-treated subclone Bx-GEM200 were left untreated (CO) or were treated with gemcitabine in concentrations from 10 to 200 nM as indicated. The viability was measured by MTT assay 72 h after treatment. (B) Cells were treated as described above followed by staining with Annexin V-FITC and PI 72 h later and evaluation by FACS-analysis. The percentage of FITC-positive cells is shown as “Apoptosis (%)”.

pression of RRM1 was analyzed by Western blot, which demonstrated a significant downregulation of RRM1 (Fig. 2C). The combined treatment of Bx-GEM200 cells with gemcitabine and siRRM1 reverted gemcitabine resistance and dramatically reduced the cell viability 72 and 96 hours after treatment, compared with the nonsense siR (Fig. 2D). RRM1 expression correlates to the level of acquired gemcitabine resistance Next, we compared the degree of gemcitabine resistance in ASANPaCa, BxPC-3, PANC-1, AsPC-1, Mia-PaCa2 and Bx-GEM200 cells with the level of RRM1 expression by performing time and dose response kinetics with gemcitabine, followed by evaluation of viability by MTT assay (Fig. S1A), and of basal RRM1 expression by qRT-

RRM1 is expressed in tissues from patients with PDA To highlight these data, we evaluated the protein expression of RRM1 in tissues of normal pancreas derived from organ donors (n = 5) or in malignant pancreas from patients with PDA, which received gemcitabine chemotherapy prior to surgery (n = 8) or not (n = 13) (Fig. 4A, Table 2). The expression of RRM1 in each sample was evaluated by immunohistochemistry and the degree of expression was determined (Fig. 4B) [22]. In non-malignant tissues, most of RRM1 positive cells were found expressed in pancreatic island, but in malignant tissue, RRM1 was expressed throughout the tumor tissues (Fig. 4C). Pancreatic islets are clusters of about 3000 to 4000 cells within acinar pancreatic tissue. The pancreatic islets contain the endocrine, hormone-producing cells to regulate the blood sugar. The islets are surrounded by a network of capillaries, which appear as clear spaces. The average expression of RRM1 in malignant tissue was higher than in non-malignant tissue (Fig. 4D), although not statistically relevant. Also, the mean expression of RRM1 was slightly higher in tissue of patients that received gemcitabine prior to surgery, which supports our former conclusion, that gemcitabine treatment induces the expression of RRM1.

Table 1 Top regulated mRNA in Bx-GEM compared to BxPC-3. Top 12 up-regulated mRNA No.

Fold

Gene name

Symbol

Chromosome

1 2 3 4 5 6 7 8 9 10 11 12

17.03 15.39 14.96 5.86 5.80 5.01 4.89 4.52 4.04 3.99 3.78 3.73

Stromal interaction molecule 1 FGF receptor activating protein 1 Ras homolog gene family, member G Claudin 1 Tripartite motif-containing 21 Leprecan-like 1 Tripeptidyl peptidase I Scavenger receptor cysteine-rich type 1 protein M160 Myeloid/lymphoid or mixed-lineage leukemia Ribonucleotide reductase M1 polypeptide Protein kinase C, delta binding protein TAF10 RNA polymerase II

STIM1 FRAG1 RHOG CLDN1 TRIM21 LEPREL1 TPP1 M160 MLLT11 RRM1 PRKCDBP TAF10

11p15.4d 11p15.4d 11p15.4d 3q28c 11p15.4d 3q28b-q28c 11p15.4c 12p13.31c 1q21.2d 11p15.4d 11p15.4c 11p15.4c

Gene name

Symbol

Chromosome

Keratin 13 G antigen 12F Annexin A10 Forkhead box Q1 G antigen 5 G antigen 12I SRY (sex determining region Y)-box 2 SRY (sex determining region Y)-box 21 Keratin 6A Myeloid-associated differentiation marker Aldehyde dehydrogenase 3 family Low density lipoprotein receptor-related protein 3

KRT13 GAGE12F ANXA10 FOXQ1 GAGE5 GAGE12I SOX2 SOX21 KRT6A MYADM ALDH3A1 LRP3

17q21.2b Xp11.23b 4q32.3e 6p25.3a

Top 12 down-regulated mRNA No. 1 2 3 4 5 6 7 8 9 10 11 12

Fold 0.063 0.098 0.112 0.119 0.141 0.156 0.160 0.161 0.177 0.183 0.183 0.187

Elements in bold point to the target gene identified in the present study.

Xp11.23b 3q26.33b 13q32.1a 12q13.13d 19q13.41b 17p11.2d 19q13.11b

P. Fan et al./Cancer Letters 373 (2016) 130–137

Fig. 2. RRM1 upregulation mediates gemcitabine resistance. (A) mRNA was harvested from BxPC-3 and Bx-GEM200 cells, followed by gene expression profiling in triplicate using the Illumina HumanHT-12 v4 Expression BeadChip. A database search in PubMed and Web of Science was performed with the terms “pancreatic cancer”, “gemcitabine” and “chemoresistance”. A heat map of candidate mRNAs was created, which combines the array and database results. The top downregulated candidate RRM1 in Bx-GEM200 cells is marked. The scale from 6 to 14 marks the intensity of differential regulation of genes: high expression (red), middle expression (black), low expression (green). (B) Upper panel: total RNA and cytoplasmic proteins were extracted from BxPC-3 and Bx-GEM200 cells and the expression of RRM1 was analyzed by qRT-PCR and Western blot. GAPDH and β-actin were used as controls. Lower panel: BxPC-3 and Bx-GEM200 cells were transferred to glass slides by cytospin centrifugation. The expression of RRM1 was detected by a specific antibody and immunofluorescence microscopy. Representative pictures are shown at 200 × magnification. The scale indicates 50 μm. (C) 100 nM RRM1 siRNA or 100 nM Allstar negative siRNA (Mock) were transfected to Bx-GEM200 cells seeded in 6 well plates in triplicate. Proteins were harvested 72 h later. The expression of RRM1 was analyzed by Western blot. (D) Bx-GEM200 cells were treated with 100 nM specific or mock siR in presence or absence of gemcitabine (100 nM). The viability was measured by MTT assay 24, 48, 72 and 96 h later. All experiments except of the gene expression analysis were performed at least three times.

miR-101-3p inhibits expression of RRM1 and directly targets RRM1 3’UTR To expand our analysis to an epigenetic impact of gemcitabineinduced expression of RRM1, we examined miR expression in BxPC-3 and Bx-GEM cells by a miR Microarray (Release 19.0), which contains 2006 different miRs. By this way, we identified 665 miRs, which were significantly up- or downregulated in Bx-GEM cells and the most significant top 12 up- and top 12-downregulated miRs are shown (Table 3). To further restrict the pool of miR candidates, we used the bioinformatics databases mirwalk, targetscan.org and microrna.org to collect all of miRs, which computationally target RRM1 (Table S2) and the resulting miR pool is shown in a heat map (Fig. 5A). Excitingly, most of the miRs, which significantly differed between BxPC-3 and Bx-GEM cells and targeted RRM1 in silico, were downregulated in Bx-GEM cells and miR-101-3p was on the top of these downregulated miRs. Therefore, putative binding sites of miR101-3p in the RRM1 3′ UTR region were analyzed by pairwise

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Fig. 3. RRM1 upregulation correlates to gemcitabine resistance. The primary human PDA cell line ASAN-PaCa and the established human PDA cell lines BxPC-3, PANC1, AsPC-1, MIA-PaCa2 and Bx-GEM200 were treated with different gemcitabine concentrations as indicated in Fig. S1A. The triangle above the diagrams of the present figure indicates the grade of resistance, based on the results obtained in the experiment shown in Fig. S1A. (A) Total RNA of each untreated cell line was harvested and the RRM1 mRNA expression was measured by qRT-PCR. The percentage of expression was set to 1 for ASAN-PaCa and the percentage of RRM1 expression in the other cell lines was related to the level in ASAN-PaCa. GAPDH were used as control. (B) In the lower panel, the protein expression of RRM1 was analyzed by Western blot and β-actin served as loading control. (C) RRM1 mRNA expression was measured by qRT-PCR in parental BxPC-3 cells or the derived subclones Bx-GEM50 (resistant up to 50 nM gemcitabine), Bx-GEM100 (resistant up to 100 nM gemcitabine), and Bx-GEM200 (resistant up to 200 nM gemcitabine). The triangle above the diagram indicates the grade of resistance (compare Fig. S1B). In the lower panel, the protein expression of RRM1 was analyzed by Western blot and β-actin served as loading control. The qRT-PCR was performed in triplicate and the experiments were performed at least three times.

sequence alignment, which resulted in the identification of 2 binding sites (Fig. 5B). Then we measured the expression of miR-101-3p by qRT-PCR, which confirmed a strong, and significant downregulation in Bx-GEM200 cells (Fig. 5C). The lipofection of miR-101-3p mimics led to a strongly enhanced expression of miR-101-3p in BxGEM200 cells after 24 and 48 hours (Fig. 5D) – simultaneously, the expression of RRM1 mRNA and protein was significantly reduced (Fig. 5E). Most importantly, the co-transfection of the plasmid pLightSwitch_RRM1_3’UTR_Renilla with miR-101-3p mimic strongly decreased the luciferase activity at 24 h, while the co-transfection of the empty LightSwitch_Renilla control vector (without RRM13’UTR) and miR-101-3p mimic alone did not change the luciferase activity (Fig. 5F), as expected. We also tried to restore the gemcitabine resistance of AsPC-1 and Panc-1 cells by lipofection of a miR-1013p inhibitor. However, we could not observe a significant reversal of gemcitabine resistance, as measured by MTT-assay (Fig. S2). Our explanation is that other miRNAs may compete and replace the function of inhibited miR-101-3p and that additional mechanisms despite miRNA-regulation may be involved in induction of gemcitabine resistance. miR-101-3p reverses gemcitabine resistance Finally, we performed time-response kinetics evaluated by MTTassay and RRM1 qRT-PCR with Bx-GEM200, PANC-1, AsPC-1 and MIA-PaCa2 cells before and after lipotransfection with miR-1013p mimics or mock mimics in the presence or absence of GEM. Whereas miR-101-3p alone strongly reduced the viability of PANC-1 and AsPC-1 cells, it had no obvious effect in Bx-GEM200 and MIAPaCa2 cells (Fig. 6A). However, the combination of miR-101-3p with gemcitabine significantly enhanced the cytotoxicity in all exam-

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ined cell lines. On mRNA level, miR-101-3p reduced the basal expression and the gemcitabine-induced expression of RRM1 in all cell lines (Fig. 6B). Discussion

Fig. 4. RRM1 is upregulated in malignant patient-derived pancreas tissue. (A) The expression of RRM1 was examined in pancreas tissue from donors with nonmalignant pancreas (normal, n = 5), or from patients with PDA, which received gemcitabine prior to surgery (GEM+, n = 8), or not (GEM-, n = 13) by immunohistochemistry. (B) The percentage of positive cells in these tissues was defined by counting the number of positive cells in 10 randomly chosen areas. The score of 0 to 4 was given according to the percentage of positive cells. (C) Representative images of each group are shown at 100 × magnification and the bar indicates 50 μm. RRM1positive cells appear dark brown and the arrows mark RRM1-positive pancreatic islets. (D) The average expression of each group is shown ±SD.

Considering the still poor prognosis of patients suffering from PDA, the detection of mechanisms underlying the pronounced therapy resistance and the development of novel therapeutic strategies is a prerequisite to achieve better outcomes. The data of the present manuscript indicate that high RRM1 expression correlates to high gemcitabine resistance. The upregulation of RRM1 directly correlated to low expression of miR-101-3p, which binds to two target sequences in the 3′ UTR of RRM1. The lipofection of miR-101-3p mimics inhibited RRM1 expression and sensitized resistant PDA cells to gemcitabine-induced cytotoxicity. In order to identify the underlying mechanisms for chemotherapy resistance, we performed a microarray profiling analysis to compare the gene expression between the gemcitabine-sensitive PDA cell line BxPC-3 and its derived gemcitabine-resistant subclone Bx-GEM. By this way we selected RRM1 as top candidate and verified the array results by detection of RRM1 overexpression in BxGEM and other gemcitabine-resistant cell lines and malignant patient tissue. The correlation of RRM1 expression to gemcitabine resistance is explained by the fact that RRM1 is the large subunit of ribonucleotide reductase, which is required for DNA synthesis [8,9]. The dCTP analog gemcitabine inhibits DNA strand synthesis [10], but a high expression of RRM1 leads to more endogenous dCTP synthesis [8], which competes with gemcitabine metabolites and thus induces gemcitabine resistance. Previous studies underline our finding of a critical role of RRM1 in chemoresistance and demonstrate its involvement in resistance toward gemcitabine in nonsmall-cell lung cancer [13], intrahepatic carcinoma [14], gastric cancer [12] and pancreatic cancer [15]. Likewise, one recent report shows an enhanced expression of RRM1 in six GEM-resistant and four highly GEM-resistant subclones derived from the pancreatic

Table 2 Characteristics of patients and PDA tumor tissues. No.

Gender

Age

Grading

GEMtreatment

RRM1 expression

Score

5008 5009 5026 5027 5031 5024 4941 4911 4730 4710 4630 4529 4409 4945 4912 4827 4794 4785 4318 4151 3738

m f m f m f m m m m f m m m m m f m m m f

64 51 74 60 60 61 58 68 76 73 68 72 72 63 54 56 61 58 63 62 71

pT3, pN1, (19/55), G2, L1, V1, Pn1 pT3, pN1, G3, R0 pT3, pN1, (8/33), G3, L1, V1 pT3, pN1, (2/24), V1, Pn1, G2, R1 pT3, pN1, (4/35), L1, V1, Pn1, G3, R1 pT3, pN1, (27/37), L1, Pn1, G3, R1 pT3, pN1, (9/21), Pn1, L1, G3, R1 pT3, pN1, (10/22), G3, R1 pT3, pN1, (5/22), L1, G2 pT3, pN1, (2/35), L1, V0, PN1 pT3, pN1, 82/25), G2, R0 pT3, pN1, (4/61), L1, G3 pT3, pN1, (9/18), G2 ypT3, ypN0, (0/7), Pn1, R1 ypT3, ypN1, yPn1, (1/18), R1 ypT3, ypN0, (0/26), L1, Pn1, R1 pT3, pN1, (1/10), G3, R1 ypT3, ypN0, (0/15), R0 ypT3, ypN0, (0/11), G0 ypT3, ypN1, (1/23) ypT3, ypN0 (0/13)

− − − − − − − − − − − − − + + + + + + + +

+ ++ ++ ++ ++ + + + + + + + + − ++ ++ + + +++ + +++

1 2 2 2 2 1 1 1 1 1 1 1 1 0 2 2 1 1 3 1 3

f: Female; m: Male; T1: Tumor limited to the pancreas, 2 cm or less in greatest dimension; T2: Tumor limited to the pancreas, more than 2 cm in greatest dimension; T3: Tumor extending beyond the pancreas; T4: Tumor involving the coeliac axis or the superior mesenteric artery; N0: No regional lymph node metastasis; N1: Regional lymph node metastasis; M0: No distant metastasis; M1: Distant metastasis; G1: Well-differentiated; G2: Moderately differentiated; G3: Poorly differentiated; pT: Primary tumor; y: pN1: Sentinel lymph node metastasis; In those cases in which classification is performed during or following multimodality therapy, the cTNM or pTNM category is identified by a y prefix; L1: Lymphatic invasion; R1: Microscopic residual tumor.

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Table 3 Top regulated miRs in Bx-GEM cells. No.

1 2 3 4 5 6 7 8 9 10 11 12

Top 12 down-regulated miRs MiR name

Fold change

hsa-miR-210 hsa-miR-486-3p hsa-miR-1246 hsa-miR-486-5p hsa-miR-455-3p hsa-miR-132-3p hsa-miR-135b-5p hsa-miR-205-5p hsa-miR-378a-3p hsa-miR-313-5b hsa-miR-30a-5p hsa-miR-378i

0.27 0.36 0.37 0.42 0.52 0.54 0.55 0.56 0.57 0.59 0.61 0.61

No.

1 2 3 4 5 6 7 8 9 10 11 12

Top 12 up-regulated miRs MiR name

Fold change

hsa-miR-885-5p hsa-miR-328 hsa-miR-3617-3p hsa-let-7d-3p hsa-miR-6511a-3p hsa-miR-449b-3p hsa-let-7b-3p hsa-miR-615-3p hsa-miR-5584-3p hsa-miR-204-5p hsa-miR-483-3p hsa-miR-197-3p

3.58 3.50 3.05 2.67 2.66 2.55 2.49 2.45 2.40 2.39 2.36 2.25

Fig. 5. miR-101-3p binds to the RRM1 3’UTR and inhibits RRM1 expression. (A) Total miRs were harvested from BxPC-3 and Bx-GEM200 cells, followed by miR expression profiling in triplicate using the Agilent miRNA Microarray (Release 19.0). A database analysis using “mirwalk”, “targetscan.org” and “microrna.org” with the key word “RRM1” was performed to select miRs related to RRM1. The heat map combines the relevant array and database results. The miRs, which were candidates in both assays were given priority and are at the top of the heatmap. The top down regulated candidate miR-101-3p is marked. The scale from 5 to 9 marks the intensity of differential miRs in BxPC-3 and Bx-GEM200 cells: high expression (red), middle expression (black) and low expression (green). (B) Putative binding sites of miR-101-3p in the RRM1 3′UTR were identified using the database “targetscan” and the resulting sequence homologies at base pairs 135–142 and 420–426 are shown. (C) Total RNA was harvested from BxPC-3 and Bx-GEM200 cells and the relative expression levels of miR-101-3p were measured by qRT-PCR. The expression levels were normalized to RNU6B and the percentage of miR-101-3p expression in BxPC-3 cells was set to 1 and the expression level in Bx-GEM200 cells was related to BxPC-3. (D) Mimics of miR-101-3p (50 nM) were lipofected to Bx-GEM200 cells. AllStar negative control siRs (50 nM) served as control (Mock). Total miRs were harvested 24 and 48 h after transfection. The level of miR-101-3p was measured by qRT-PCR. (E) Bx-GEM200 cells were lipofected as described above. Total mRNAs were harvested 24 and 48 h after transfection and total proteins were harvested 72 h later. RRM1 mRNA and protein levels were analyzed by qRT-PCR and Western blot. GAPDH and β-actin served as controls for equal conditions. (F) Dualluciferase reporter assay. The wild type RRM1 3′-UTR cloned into a pLightSwitch renilla plasmid or empty vector (0.5 ng/μl) were lipofected into Bx-GEM200 cells in the presence or absence of 50 nM miR-101-3p mimics. The co-transfection of a firefly luciferase plasmid (0.25 ng/μl) served as control for equal conditions. The expression of Renilla and firefly luciferase was detected on a FLUOstar Omega microplate reader. Renilla luciferase activities were normalized to firefly luciferase activities. All assays were performed in triplicate and repeated at least three times (mean ± SD).

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terestingly, we found a selective expression of RRM1 in pancreatic islands of non-malignant pancreas tissues, whereas the expression of RRM1 in malignant tissue was not restricted to specific cells. As a logical consequence, the relationship between RRM1-derived resistant pancreatic cancer cells and pancreatic islet cells may be suspected, although the underlying function of RRM1 in islet cells is unclear and needs further investigation. We performed a miR array analysis and identified by a subsequent computational analysis miR-101-3p as top downregulated candidate, which might be responsible for the observed enhanced RRM1 expression and chemoresistance in Bx-GEM cells. This hypothesis was confirmed by lipofection of miR-101-3p mimics, which inhibited RRM1 expression and thereby sensitized for gemcitabineinduced cytotoxicity. This finding is new and was unknown so far. What was known is a function of miR-101-3p in inhibition of CSC characteristics via repression of EZH2 in pancreatic cancer [23] and a reversal of cisplatin resistance by interaction with autophagy in osteosarcoma and hepatocellular carcinoma [24,25]. However, although we demonstrated a direct binding of miR-101-3p to two binding sites in the RRM1 3’UTR, the lipofection of a miR-101-3p mimic was not as potent in reversal of gemcitabine resistance as the direct inhibition of RRM1 expression by a siRNA approach. Also, the lipofection of a miR-101-3p inhibitor did not reverse the gemcitabine resistance in AsPC-1 and Panc-1 cells. We assume that additional miRs are involved in regulation of RRM1 expression and that despite of RRM1 other mechanism involved in regulation of gemcitabine resistance. Though the expression level of miR-1013p was not confirmed in tissue from patients with PDA in the present study, a recent report detected the expression of miR-101-3p in clinical pancreas specimens, where the level of miR-101-3p expression was higher in non-invasive IPMNs and normal tissues compared with invasive IPMNs [26]. Likewise, a decreased miR-101-3p expression was observed in malignant tissue of patients suffering from PDA and correlated to a significantly poor prognosis [15]. In conclusion, our study demonstrates that long-term treatment of PDA cells with gemcitabine induced pronounced therapy resistance. The RRM1 gene is a major mediator of resistance and its expression is regulated by direct binding of miR-101-3p to two binding sites in the RRM1 3’UTR. The overexpression of miR-1013p mimics inhibited the expression of RRM1 and partially reversed gemcitabine-resistance. Acknowledgement

Fig. 6. miR-101-3p sensitizes chemoresistant PDA cells to gemcitabine. (A) Fifty nanomolar of miR-101-3p mimics or negative miR (Mock) were lipofected to BxGEM200, PANC-1, AsPC-1 and MIA-PaCa2 cells in presence or absence of gemcitabine (100 nM). The viability was measured by MTT assay 24, 48, 72 and 96 h later. (B) Total RNA was harvested 24 h after treatment and the expression level of RRM1 was measured by qRT-PCR and analyzed as described in Fig. 3A. The MTT assays were performed in octuplicate and the qRT-PCRs in triplicate. The experiments were repeated three times and the means ± SD are shown.

cancer cell line BxPC-3 [18]. Similarly, Nakahira et al. [15] demonstrated that the level of RRM1 mRNA expression in the established PDA cell lines PSN1, MiaPaCa2, BxPC-3, Panc1 and PCI6 correlated to the level of gemcitabine resistance. Our study extends these results by the use of several established and primary cell lines and patient tissue and the use of siRNA-mediated inhibition of RRM1, which restored chemosensitivity. As well, RRM1 siRNA was used recently in established cell lines from human non-small-cell lung cancer and murine lung cancer to inhibit gemcitabine resistance [16,17]. In-

We thank Dr. Svetlana Karakhanova and Zhuo Yue for critical text revision and the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ) for providing the Illumina Whole-Genome Expression Beadchips and related services. We thank the PancoBank of our clinic (Prof. M. W. Büchler) for the collection and processing of pancreas specimens supported by the team of the European Pancreas Center (E. Soyka, S. Bauer, A. Hieronymus, B. Bentzinger, K. Ruf) and funded by the Heidelberger Stiftung Chirurgie, the Federal Ministry of Education and Research (BMBF 01GS08114) and the Biomaterial Bank Heidelberg/BMBH (Prof. Dr. P. Schirmacher; BMBF 01EY1101). Our study was supported by grants from the German Cancer Aid (Deutsche Krebshilfe 109362, 111299), Federal Ministry of Education and Research (BMBF 031A213), Heidelberger Stiftung Chirurgie, Stiftung für Krebs und Scharlachforschung, Dietmar Hopp-Stiftung and Hanns A. Pielenz Stiftung. The first author Fan Pei was supported by a stipend from the China Scholarship Council (File No. 201308080097). Conflict of interest None of the authors has a conflict of interest to disclose regarding the publication of the present manuscript.

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