ERK signaling pathways

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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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

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Q1 Q2 Original Articles

MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways Q3 Yanmin Xu, Ji Huang, Leina Ma, Juanjuan Shan, Junjie Shen, Zhi Yang, Limei Liu,

Yongli Luo, Chao Yao, Cheng Qian * Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China

A R T I C L E

I N F O

Article history: Received 7 October 2015 Received in revised form 23 November 2015 Accepted 27 November 2015 Keywords: miR-122 IGF-1R Sorafenib Drug resistance Hepatocellular carcinoma

A B S T R A C T

Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVPAEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance. © 2015 Published by Elsevier Ireland Ltd.

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Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and is a leading cause of cancer death worldwide [1,2]. Until recently, the overall survival rate of HCC patients was very poor due to late-stage of detection and unavailability of effective drugs [3,4]. Molecular targeted therapy, which aims to correct specific molecular derangements in cancer cells or their microenvironment, is currently the standard treatment for patients with advanced HCC [5]. Sorafenib (Nexavar) is the first and only targeted therapy to clinically improve overall survival in patients with advanced HCC, and has given hope to researchers searching for effective agents to combat HCC [6–8]. However, most patients who initially responded to treatment with sorafenib relapsed, suggesting that chronic treatment with sorafenib is associated with development of drug resistance [9–11]. Several mechanisms are involved in the acquired resistance to sorafenib, such as crosstalk involving the PI3K/Akt and JAK-STAT pathways, hypoxia-inducible pathways, epithelial-mesenchymal transition, and others [12]. Currently, the detailed mechanism by which this phenomenon occurs remains unknown.

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* Corresponding author. Tel.: +86 23 68765957; fax: +86 23 68752247. E-mail address: [email protected] (C. Qian).

Drug resistance is a common problem associated with chronic treatment with anticancer drugs [13,14]. MicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that play important roles in the control of numerous biological processes [15–17]. Certain miRNAs impart drug resistance [18,19] or sensitivity to cancer cells [20,21]. miR-122 is the most abundant miRNA in the liver, accounting for approximately 70% of the total miRNA population [22,23]. miR-122 is frequently down-regulated in primary HCC [24]. The marked decrease in this liver-specific miRNA in HCC suggests that it might play a role in maintaining hepatic functions and emphasizes the importance of miR-122 in liver homeostasis [25]. Functional studies elucidating the tumor suppressor function of miR-122 are based on cancer cells in culture or ex vivo in mice. These studies have examined clonogenic survival, anchorage-independent growth, migration, invasion, epithelial-mesenchymal transition, and the ability to form tumors in nude mice [26,27]. However, all of these findings do not sufficiently explain the tumor suppressor potential of miR-122 in the context of drug tolerance. Our previous study showed that restoration of liver-specific miR-122 expression is involved in the regulation of multidrug resistance (MDR)-related gene expression and modulating the chemosensitivity of HCC cells [28]. We were curious to examine whether miR-122 potentiates the sensitivity of HCC cells to sorafenib.

http://dx.doi.org/10.1016/j.canlet.2015.11.034 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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In the present study, we established sorafenib-resistant cell models to further investigate the association between miR-122 expression and sorafenib resistance. Our miRNA expression array analysis revealed that miR-122 was down-regulated in acquired sorafenib resistance. Then, we used sorafenib-sensitive and -resistant cell models to comprehensively investigate the role of miR-122 in acquired resistance to sorafenib. At the same time, our gene expression profiles showed that insulin-like growth factor 1 receptor (IGF-1R) was up-regulated in sorafenib-resistant cells compared with their parental counterparts. We further confirmed that IGF-1R was a target of miR-122. Furthermore, we demonstrated that miR-122 and IGF-1R per se can be critical determinants of the occurrence of drug tolerance. These data suggest that targeting miR-122 or IGF1R may improve therapeutic benefit to HCC patients receiving sorafenib treatment.

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Materials and methods

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Tissue samples, cell culture and pharmacologic agents

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Fresh tumor specimens were obtained with informed consent from all patients according to protocols approved by the Institutional Review Board of the Southwest Hospital, Third Military Medical University. All patients underwent surgical resection of primary HCC at the Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University. Human HCC cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO-BRL), 100 U/mL of penicillin sodium and 100 mg/mL of streptomycin sulfate (Invitrogen Life Technologies, Gaithersburg, MD), at 37 °C in a 5% CO2 atmosphere. Sorafenib (Nexavar) was kindly provided by Dr. Feng Xia (Third Military Medical University, Chongqing, China). For in vitro experiments, the drugs were dissolved in DMSO, and the final concentration of DMSO was kept below 0.1%. The IGF-1R inhibitors NVP-AEW541 and picropodophyllin (PPP) were purchased from Novartis. NVP-AEW541 was dissolved in tartaric acid (25 mmol/L). PPP was dissolved in DMSO (0.5 mM). IGFI and IGFII were purchased from R&D Systems (Minneapolis, MN).

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Drug-sensitive cells were treated with sorafenib at the concentration of 10 μM for three rounds, each lasting 72 h. Viable cells remaining attached to the dish at the end of the third round of drug treatment were considered to be sorafenibresistant and were collected for experiments. Resistant cell lines were maintained in the continuous presence of 10 μM sorafenib, supplemented every 72 h.

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To confirm the functional activity of P-gp in sorafenib-resistant cells, we used the exclusion of the fluorescent dye rhodamine 123 (Rho123) as an index for P-gp activity. Rho123 is a substrate of P-gp, so inhibition of P-gp activity results in increased intracellular Rho123, reflected by higher fluorescent signals. The cells (3.0 × 105/well) were incubated in six-well plates to allow attachment overnight. Then, 5 μM Rho123 was added to the medium and incubated for 30 min at 37 °C with 5% CO2. The complete medium was removed, and the cells were washed with PBS and cultured in fresh complete medium for 30 min. Next, 5 × 105 cells were collected, rinsed once with cold PBS, and re-suspended in 500 μL cold PBS. Flow cytometry analysis of Rho123 fluorescence was performed with the BD FACS AriaII (Becton Dickinson, San Jose, CA, USA) at 585 nm.

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The 3′-UTR sequence of IGF-1R predicted to interact with miR-122 or a mutated sequence within the predicted target sites was synthesized and inserted into the SpeI and HindIII sites of the pMIR-REPORT Luciferase vector (Ambion, Austin, TX). These constructs were named pMIR-IGF-1R-3′UTR-C(Conserved)-wt or pMIR-IGF1R-3′UTR-Cmut, pMIR-IGF-1R-3′UTR-PC(Poor Conserved)-wt or pMIR-IGF-1R-3′UTRPCmut, respectively. For the reporter assay, Huh7 cells were plated onto 24-well plates and transfected with the above constructs and miR-122 mimics or miRNA control using the Effectene® Transfection Reagent (Qiagen, Hilden, Germany). A pMIRREPORT β-gal control plasmid vector (Ambion, Austin, TX) was co-transfected to normalize the difference in the transfection efficiency. After 48 h, the cells were harvested and assayed using the dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s instructions. The relative luciferase activity was calculated as the ratio between the Renilla and firefly luciferase activities. Results were obtained from three independent experiments performed in duplicate. The primer sequences are listed in the Supplemental Experimental Procedures.

RNA extraction and miRNA microarray analysis

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Total RNA including the miRNA fraction was isolated from PLC, PLC-DR3, Huh7 and Huh7-DR3 cell lines using the miRNeasy Mini Kit (QIAGEN, Valencia, CA). After extraction, the quality of each RNA sample was assessed using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA) according to the manufacturer’s instructions to ensure an RNA integrity number > 7. RNA samples with RIN ≥ 6.0 and 28S/ 18S ratio > 0.7 were used for the Agilent miRNA Chip. miRNA microarray profiling was performed using Agilent miRNA array 18.0 (ShanghaiBio Corporation, Shanghai, China) to identify candidate miRNAs expressed differently between the sorafenibresistant cells and their parental counterparts. RNA labeling and hybridization were performed using a Human miRNA Microarray and a miRNA Complete Labeling and Hyb Kit (Agilent Technologies). After washing with Gene Expression Wash Buffer, the slides were scanned with an Agilent Microarray Scanner and analyzed by GeneSpring GX software.

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mRNA microarray analysis

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Total RNA was extracted from Huh7 and Huh7-DR3 cells using TRIzol reagent (Invitrogen, Merelbeke, Belgium), and RNA was isolated with the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. RNA concentration was assessed with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), and RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). Gene expression profiling of two samples was performed by the Affymetrix Human Genome U133 Plus 2.0 array platform (CapitalBio Corporation, Beijing, China). Data were read using QuantArray R software and analyzed using Cluster3.0 and SAM2.0 software. After normalization against the control gene (GAPDH), a gene was designated as differentially expressed if its expression in sorafenib-resistant cells was ≥1.5-fold that of parental cells.

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In vitro self-renewal assays

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For sphere formation efficiency assay, the single cell was sorted and plated into ultra-low attached 96-well plates (Corning). The cells were cultured in DMEM/F12 (Sigma) with B27 (Gibco), 20 ng/mL EGF (Peprotech), 20 ng/mL bFGF (Peprotech) 10 ng/ mL HGF (Peprotech) and antibiotics. Cells were incubated at 37 °C with 5% CO2 and the number of spheres was counted after 2 ± 3 weeks.

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Cloning efficiency assays

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For colony formation, cells in single-cell suspension were plated and grown in 24-well plates at a density of 100 per well for 24 h. Cells were then treated or untreated with sorafenib or pharmacologic agents (IGFI, IGFII, NVP-AEW541 and PPP). The medium was replaced every 3 days with fresh medium containing the corresponding agents. The plates were further incubated for 14 days at 37 °C until colonies were visible. The colonies were fixed 15 min with 4% paraformaldehyde and stained with 0.1% crystal violet. The numbers of colonies were counted to assess the viability.

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Quantitative real-time PCR analysis for miRNA and mRNA expression

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For miRNA detection, reverse transcription reactions were performed by TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Real-time PCR was performed using a standard TaqMan® PCR kit protocol on a CFX96™ Real-Time PCR System (BioRad Laboratories, Hercules, CA, USA). The relative expression of the miRNAs was calculated using the -ΔΔCT method, and relative miRNA levels were normalized to U6 small non-coding RNA. For mRNA detection, first-strand cDNA synthesis and amplification were performed according to the protocol of the PrimeScript™ RT Reagent Kit (Perfect RealTime) (TaKaRa, #RR047A). Real-time PCR was completed as prescribed by the SYBR Premix Ex Taq (TaKaRa, #RR420A) with a Bio-Rad CFX96™ Real-Time PCR System. RT-PCR primers are listed in Supplemental data (Table S3). GAPDH was used for normalization.

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Bioinformatics method

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Generation of drug-resistant cells

Rhodamine 123 efflux assay

Luciferase reporter assay

We used Target Scan Release 6.2 (http://www.targetscan.org) to predict miR122 target genes.

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Western blotting analysis

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Whole-cell lysates were generated using RIPA lysis buffer (Thermo). Total protein samples were separated using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. The primary antibodies used were as follows: Sox2 (1:1000, Abcam, ab75485), Nanog (1:1000, Abcam, ab109250), IGF-1R (1:1000, Cell signaling, #9750), p-IGF-1R (pYpY1135/1136) (1:500, Novex by life technologies, #701067), RAF1(1:1000, Abcam, ab32025), AKT1 (1:1000, Abcam, ab32505), p-AKT1 (S473) (1:1000, Abcam, ab81283), ERK1/2 (1:5000, Abcam, ab54230), p-ERK1/2 (1:1000, Abcam, ab32538), STAT3 (1:1000, Abcam, ab32500), p-STAT3 (Y705) (1:1000, Abcam, ab76315), and

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Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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GAPDH (1:1000, Cell Signaling, #2118). The membrane was incubated with primary antibody at 4 °C overnight followed by horseradish peroxidase-conjugated secondary antibody the next day for 2 h at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence with ECL reagents (Pierce, Rockford, IL, USA). A GAPDH antibody was used as a loading control.

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All procedures for animal experiments were approved by the committee on the Use and Care on Animals (The Third Military Medical University, Chongqing, China) and performed in accordance with the institution guidelines. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy. Different numbers of Huh7 and Huh7-DR3 cells were counted and then re-suspended in serum-free medium that contained 50% Matrigel (BD Biosciences, Bedford, MA, USA). The cells were subcutaneously injected into male 3- to 5-week-old NOD/SCID mice. Tumor formation was evaluated regularly after injection by palpation of injection sites. The tumor weights and tumor volume were measured.

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Male 3- to 5-week-old NOD/SCID mice were subcutaneously inoculated with Huh7-IGF-1R-OE cells or Huh7-control cells (1 × 106) in serum-free medium containing 50% Matrigel (BD Biosciences, Bedford, MA, USA). Tumor formation was evaluated regularly after injection by palpation of injection sites. The different tumors were treated with sorafenib 60 mg per kg bw per day. All treatments were administered via gavage for 2 weeks. Tumor weights and volume were calculated.

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Statistical analysis

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All data are presented as means ± SD. Statistical analyses were performed by using GraphPad Prism version 5 for Windows (GraphPad Software). The differences in means between two groups were analyzed with the two-tailed unpaired Student’s t-test, and p < 0.05 was considered statistically significant.

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Tumor formation assay

Results

In vivo sorafenib gavages experiments

Chronic treatment with sorafenib leads to drug resistance in HCC cells, and sorafenib-resistant cells exhibit the properties of cancer stem cells To investigate the relationship between miRNA expression and sorafenib resistance, we established sorafenib-resistant cell models by chronic exposure to sorafenib in HCC cells. We observed that the majority of cells were killed within a few days at the highest achievable clinical concentration (10 μM) (Supplementary Fig. S1A). Accordingly, we established four different lines of sorafenib-resistant cells as described previously (Supplementary Fig. S1B) [29]. Our data showed that sorafenib-resistant cells required higher doses of the drug for partial growth inhibition than their parental cells (Supplementary Fig. S1C), and these cells grew more slowly than their parental cells (Supplementary Fig. S1D). Analysis of MDR-related gene expression

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Fig. 1. Sorafenib-resistant cells exhibit the properties of cancer stem cells. (A) Colony-formation efficiency of Huh7 and Huh7-DR3 cells (left) and T1115 and T1115-DR3 cells (right). (B) Sphere-formation efficiency of the Huh7 and Huh7-DR3 cells (left) and T1115 and T1115-DR3 cells (right). (C) RT-PCR analysis of pluripotency-associated genes Nanog, Sox2, Oct4, c-Myc, Klf4 and CD133 in drug-tolerant cells versus their parental counterparts. All experiments were performed in triplicate. Data are presented as the mean ± standard deviation (n = 3). (D) Immunofluorescence analysis of CSC marker expression in sorafenib-resistant cells and parental cells. (E) Western blot analysis of Nanog and Sox2 in sorafenib-resistant cells and parental cells. (F, G). The transplantation assay of Huh7 parental cells and sorafenib-resistant cells that were subcutaneously injected into NOD/SCID mice. The tumor volume and tumor weight were compared.

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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Fig. 2. miRNA microarray analysis of significantly up- and down-regulated genes in sorafenib-resistant cells compared with their parental counterparts. (A) miRNA microarray results show differentially expressed genes in sorafenib-resistant cells compared with their parental cells. The scale bar on top indicates relative levels, with red corresponding to high expression and green to low expression. (B) Compared with sorafenib-sensitive cells, 31 miRNAs were up-regulated and 30 miRNAs were down-regulated in PLC- and Huh7-sorafenib-resistant cells. (C) Heat map displaying 61 miRNAs with 1.5-fold or more differential expression between the sorafenib-resistant cells versus their parental counterparts. (D) RT-PCR verified miR-122 expression in sorafenib-resistant cells and their parental counterparts.

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found that MDR1 expression was increased more than the other four MDR genes in sorafenib-resistant cells versus their parental counterparts (Supplementary Fig. S1E and F). P-gp activity was also statistically higher in sorafenib-resistant cells, compared with parental cells (Supplementary Fig. S1G). These data indicate that we successfully established sorafenib-resistant cell models. Chemotherapeutic agents can enrich cancer stem cells (CSCs), and liver CSCs are resistant to sorafenib treatment [30,31]. Thus, we investigated whether sorafenib-resistant cells exhibited characteristics of CSCs. Our data showed that sorafenib-resistant cells had higher colony and sphere formation efficiencies compared with their parental counterparts (Fig. 1A and B). Increased Nanog, c-Myc, Klf4, Oct4, Sox2, and CD133 were observed in sorafenib-resistant cells compared with their parental counterparts at the mRNA and protein levels (Fig. 1C, D and E). Furthermore, sorafenib-resistant cells formed xenograft tumors more efficiently in NOD/SCID mice than their parental counterparts (Fig. 1F). These data indicate that sorafenibresistant cells exhibited the properties of CSCs.

sorafenib-resistant cells was markedly different from that of parental cells (Fig. 2A). Hierarchical clustering analysis of miRNA expression showed significant changes (>1.5-fold difference) in expression for 61 out of 940 (6.49%) human miRNAs between sorafenib-resistant PLC and Huh7 cells and their parental cells (Fig. 2B and C, Table S1). Of these miRNAs, 31 miRNAs were up-regulated and 30 miRNAs were down-regulated in sorafenib-resistant cells. Our miRNA microarray data revealed that miR-122 expression was significantly reduced in sorafenib-resistant cells. We verified that the expression of miR122 was reduced in sorafenib-resistant cells by RT-PCR (Fig. 2D). These data are consistent with our previous reports indicating that miR122 expression is significantly reduced in HCC and restoration of miR122 could re-sensitize cancer cells to chemotherapeutic agents [28,32]. More importantly, liver-specific miR-122 plays a pivotal role in maintaining hepatic functions [25], and sorafenib-resistant, ADM- or VCRchemoresistant cancer cells all display the same target downregulation. These collective findings imply that miR-122 may be a promising predictive biomarker for drug tolerance in HCC.

Liver-specific miR-122 expression is significantly reduced in sorafenib-resistant cells

Overexpression of miR-122 renders sorafenib-resistant cells sensitive to sorafenib via induction of cell apoptosis

To determine whether miRNAs are responsible for sorafenib resistance, we carried out a miRNA microarray in sorafenib-resistant cells and their parental cells. The pattern of miRNA expression in

As miR-122 expression was down-regulated in sorafenib-resistant cells, we next investigated the role of miR-122 in these cells. Infection of adenovirus expressing miR-122 or transfection with

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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Fig. 3. In HCC sorafenib-resistant cells, up-regulated miR-122 inhibits cell viability and induces apoptosis. (A, B) Huh7-DR3 and PLC-DR3 cells were infected with adenovirus at the MOI of 5 (A) or transfected with miRNA mimics at 200 nM (B). miR-122 expression was measured 24 h after transfection or infection. (C, D) Huh7-DR3 and PLC-DR3 cells were treated with Ad-miR-122, NC or sorafenib alone or the combination of Ad-miR-122 or NC with sorafenib (C); or sorafenib-resistant cells were transfected with miRNA mimics, NC or sorafenib alone or the combination of miR-122 mimics or NC with sorafenib (D). Cell viability was determined by CCK-8 assay at different time-points after treatment. Data are presented as means ± SD from three independent experiments. (E) Huh7-DR3 and PLC-DR3 cells were treated by sorafenib (10 μM), Ad-miR-122 (5 MOI), NC (5 MOI) or the combination of sorafenib with Ad-miR-122 or NC. Cells were treated for 24 h and were analyzed for apoptotic rate by flow cytometry. The experiment was performed in triplicate, and representative image are shown.

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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miR-122 mimics resulted in high expression of miR-122 in sorafenibresistant cells (Fig. 3A and B). However, infection of adenovirus expressing miR-122 or transfection with miR-122 mimics in sorafenibresistant cells did not induce cytotoxicity to these cells. Treatment of sorafenib-resistant cells with sorafenib at 5 μM or 10 μM alone only induced mild cytotoxicity. In contrast, treatment of sorafenibresistant cells either infected with adenovirus expressing miR-122 or transfection with miR-122 mimics with sorafenib induced dramatic cytotoxicity (Fig. 3C and D; Supplementary Fig. S2A and B). These results indicate that significant antitumor activity could be achieved by the combination of miR-122 overexpression and sorafenib treatment in sorafenib-resistant cells. We further explored whether antitumor activity induced by miR122 and sorafenib in sorafenib-resistant cells was due to induction of apoptosis. Our data showed that the combined treatment with Ad-miR122 and sorafenib increased the apoptotic rate to 18.49% and 22.23% in Huh7 and PLC-sorafenib-resistant cells, respectively, which were significantly higher than Ad-miR122 or sorafenib treatment alone (Fig. 3E, Supplementary Fig. S3A and B). All together, these results suggest that induction of apoptosis is a major mechanism by which miR-122 modulates the sensitivity of sorafenib-resistant cells to sorafenib.

miR-122 expression is significantly reduced in sorafenib-resistant cells and negatively regulates the expression of IGF-1R miRNAs function as negative regulators of the expression of protein-coding genes and play critical roles in pathogenesis of cancers. Thus, miRNA target prediction algorithms were used to predict miR-122 target genes, and 172 conserved targets were predicted. We compared the expression of these 172 conserved targets between sorafenib-resistant cells and parental cells using the Affymetrix platform, focusing on genes whose expression was significantly up-regulated in sorafenib-resistant cells. Our results showed that 15 targets satisfied this criterion (Table S2). Then, we verified the expression of these targets by RT-PCR. Only IGF-1R expression was markedly increased in Huh7 sorafenib-resistant cells; the expression of the other 14 target genes did not change significantly (Fig. 4A). IGF-1R was also significantly up-regulated in PLC sorafenib-resistant cells (Fig. 4B). The increased protein level was validated for IGF-1R in sorafenib-resistant cells by western blotting (Fig. 4C). We next attempted to confirm whether IGF-1R was a true target of miR-122. For this purpose, a luciferase reporter assay was used in sorafenib-resistant cells transfected with pMIR-IGF1R-3′UTR-CW (conserved wild type) or pMIR-IGF-1R-3′UTR-Cmut

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Fig. 4. IGF-1R is a functional target of miR-122 and is involved modulating the sorafenib sensitivity of HCC drug-tolerant cells. (A) RT-PCR analysis of miR-122 target expression in Huh7-DR3 and Huh7 cells. (B). The expression of IGF-1R in PLC-DR3 and PLC cells. (C) Western blot analysis of IGF-1R in PLC- and Huh7-sorafenib-resistant cells and their parental cells. (D) The conserved miR-122 cognate site in 3′-UTR of IGF-1R. (E). Verification of the conserved miR-122 cognate site in 3′-UTR of IGF-1R. Luciferase activity driven by 3′-UTR of IGF-1R was inhibited by ectopic expression of miR-122. Huh7 cells were co-transfected with firefly luciferase-3′-UTR-(IGF-1R) or 3′UTR of IGF-1R with the miR-122 complementary site deleted and miR-122 mimics or control RNA (100 nM) along with a control plasmid using Effectene® Transfection Reagent. Reduction of luciferase activity driven by IGF-1R-3′UTR-wt was observed (left panel). The IGF-1R-3′UTR-Mutant construct failed to suppress the luciferase activity (right panel). (F, G) Huh7-DR3 (F) and PLC-DR3 (G) cells were treated with sorafenib, sorafenib plus IGFI, or sorafenib plus IGFII for 24 h and were analyzed for apoptotic rate. The untreated group was considered as control. The experiment was performed in triplicate, and representative image are shown.

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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Fig. 5. Analysis of apoptosis in Huh7 parental cells and drug-tolerant cells. (A) Huh7 parental cells were incubated with different concentrations of sorafenib for 48 h, and the apoptotic rate was measured by flow cytometry. (B) Huh7 parental cells and drug-tolerant cells were treated with 10 μM sorafenib for 48 h, and the cell apoptotic rate was measured. (C) Drug-tolerant cells were treated with sorafenib (5 μM), PPP (0.01 μM), or NVP-AEW541 (2 μM) alone or the combination of sorafenib with IGF-1R inhibitor (PPP or NVP-AEW541), and the percentage of apoptotic cells was detected after 24 h. (D) Huh7 parental cells were treated by sorafenib (10 μM), IGFI (250 ng/mL), or IGFII (500 ng/mL) alone or the combination of sorafenib with IGFI or IGFII for 24 h and were analyzed for cell apoptotic rate. The experiment was performed in triplicate. Data are presented as the mean ± standard deviation (n = 3).

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(conserved mutant type) along with miR-122 mimics or control RNA (Fig. 4D; Supplementary Fig. S4A). A significant decrease of luciferase activity was observed only in the cells transfected with pMIRIGF-1R-3′UTR-CW and miR-122 mimics, but not in the cells transfected with pMIR-IGF-1R-3′UTR-Cmut and miR-122 mimics (Fig. 4E; Supplementary Fig. S4B). These data indicate that miR122 negatively regulates IGF-1R expression by interacting with its 3′-UTR. Furthermore, we explored whether induction of cell apoptosis by up-regulation of miR-122 is related to IGF-1R, a tyrosine kinase receptor with high affinity for insulin-like growth factor I (IGFI) and IGFII. Our results showed that the increased apoptotic rate induced by overexpression of miR-122 was reversed by either IGFI or IGFII in sorafenib-resistant cells (Fig. 4F and G; Supplementary Fig. S4C and D). Together, these data suggest that IGF-1R is a functional target of miR-122. IGF-1R activation can confer HCC cells resistance to sorafenib and induce anti-apoptotic effects in drug-tolerant cells IGF-1R activation has been linked to drug resistance by protection of cells from a variety of apoptosis-inducing agents [33–35]. To investigate whether IGF-1R plays a critical role in sorafenib-

resistant cells, we first detected the apoptotic effects of sorafenib on parental cells. Our data showed that sorafenib could induce apoptosis of parental cells in a dose-dependent manner (Fig. 5A; Supplementary Fig. S5A). However, sorafenib at the concentration of 10 μM, which induced a high apoptotic rate in parental cells, and did not cause obvious apoptosis in sorafenib-resistant cells (Fig. 5B; Supplementary Fig. S5B). To further investigate whether IGF-1R is required for the anti-apoptotic effect in sorafenib-resistant cells, we treated drug-tolerant cells with sorafenib combined or not with an IGF-1R inhibitor and analyzed the apoptotic rate. As shown in Fig. 5C (Supplementary Fig. S5C) treatment of drug-resistant cells with PPP or NVP-AEW541 combined with sorafenib dramatically increased the percentage of the apoptotic population from 9.95% (sorafenib) to 58.34% (sorafenib and PPP) or 38.18% (sorafenib and NVPAEW541). These results demonstrated that PPP or NVP-AEW541 can abrogate IGF-1R activation and counteract IGF-1R’s protective effect on sorafenib-resistant cells. On the other hand, to show whether the activation of IGF-1R is sufficient to be obtained the ability of anti-apoptotic in parental cells, we treated parental cells with IGFI or IGFII to activate IGF-1R signaling. The results indicated that IGFI or IGFII combined with sorafenib resulted in a notable decrease in the apoptotic rate from 19.68% (sorafenib) to 6.70% (sorafenib and

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IGFI) or 7.87% (sorafenib and IGFII) (Fig. 5D; Supplementary Fig. S5D). Our data suggest that IGF-1R activation potentially contributes to sorafenib resistance by inhibiting cell apoptosis. IGF-1R leads to induction of pro-survival signals and mediates RAS/ RAF/ERK pathway in sorafenib-resistant cells IGF-1R has gained increasing attention as a promising target in cancer therapy, but its role as a critical determinant of sorafenib resistance and therapeutic target in HCC has not been systematically explored. The serine/threonine protein kinase Akt is one of the most frequently hyperactivated protein kinases in human cancer. Hyperactivation of Akt is associated with resistance to apoptosis, increased cell growth, cell proliferation and cell energy metabolism [36]. Akt has emerged as a critical downstream effector of IGF1R. However, the mechanism by which IGF-1R activates Akt is not fully understood in sorafenib-resistant cells. To elucidate the molecular mechanisms underlying acquired resistance to sorafenib, we analyzed the effect of sorafenib on activation of IGF-1R and the downstream RAS/RAF/ERK signaling pathway in both parental and drug-tolerant cells. We first evaluated both the expression of IGF1R and phosphorylation of IGF-1R at pYpY1135/1136, which is indicative of kinase activation. We found that IGF-1R remained phosphorylated in drug-resistant cells compared with their parental counterparts (Fig. 6A). IGF-1R can activate both the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and the Ras/Raf/ mitogen-activated protein (MAP) kinase pathway [37]. Considering that the IGF-1R and PI3K/AKT pathways play important roles in mediating cell survival, we examined the effect of IGF-1R activation on the expression of some components of the RAS/RAF/ERK signaling pathways that are known to be important for survival. Our data showed that PLC-DR3 and Huh7-DR3 cells expressed high levels of phospho-AKT1, phospho-ERK1/2, phospho-STAT3 and RAF1 but had an inhibition effect on AKT1 and ERK1/2, which represented signaling activation (Fig. 6B). To further show a critical role for IGF1R in RAS/RAF/ERK activation, we determined whether suppressing the expression of IGF-1R could lead to inhibition of RAF1, AKT1, ERK1/2 phosphorylation. Treatment with PPP or NVP-AEW541 significantly reduced RAF1 and phosphorylated ERK1/2 in sorafenibresistant cells (Fig. 6C and D). However, it had no effect on AKT1 activation (data not shown). Taken together, these results support a key role for IGF-1R activation in mediating ERK1/2 phosphorylation through the RAS/RAF/ERK kinase pathway, which could represent a mechanistic explanation of resistance to sorafenib.

Fig. 6. IGF-1R mediates PI3K signaling in sorafenib-resistant cells. (A) Expression and phosphorylation of IGF-1R was assessed in PLC- and Huh7-sorafenib-resistant cells and their parental counterparts. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Expression of RAF1, AKT1, p-AKT1, ERK1/2, p-ERK1/ 2, STAT3, and STAT3-PY705 were assessed in PLC- and Huh7-sorafenib-resistant cells and their parental cells. (C, D) Huh7-sorafenib-resistant cells were treated with increasing concentrations of PPP (0.01 μM or 0.1 μM) or NVP-AEW541 (2 μM or 10 μM). The effects of IGF-1R inhibition on RAF1, ERK1/2, p-ERK1/2, AKT1, and p-AKT1 were assessed by immunoblotting.

Inhibition of IGF-1R activity disrupts tolerance to sorafenib, and IGF1R signaling is required for the emergence of drug resistance To test a specific requirement for IGF-1R in sorafenib tolerance, we depleted IGF-1R expression in sorafenib-resistant cells using RNA interference. In sorafenib-resistant cells, stable knockdown of IGF1R led to significantly increased sensitivity of cancer cells to sorafenib (Fig. 7A), and ablation of IGF-1R also markedly reduced the yield of resistant colonies following sorafenib treatment (Fig. 7B). Consistent with our results from RNA interference, treatment with IGF1R inhibitor (PPP or NVP-AEW541) together with sorafenib in drugtolerant cells virtually eliminated the emergence of drug-tolerant colonies (Fig. 7C). IGF-1R inhibitor alone only partially inhibited the emergence of drug-resistant colonies (Fig. 7C). Furthermore, activation of IGF-1R by IGFII increased drug-resistant colonies in parental cells (Fig. 7D). Overexpression of IGF-1R formed xenograft tumors in NOD/SCID mice that were resistant to sorafenib as administered by direct sorafenib gavage (60 mg/kg/day) for 2 weeks compared with control groups (Fig. 7E). The collective findings revealed that a subpopulation of drug-tolerant cells that transiently exhibited a distinct phenotype characterized by the engagement of

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IGF-1R activity. Substantial efforts to elucidate the molecular basis for such drug resistance have revealed a variety of mechanisms, including drug efflux, acquisition of drug binding-deficient mutants of the target, and engagement of alternative survival pathways [38]. We still have good reason to believe that IGF-1R plays a pivotal role in sorafenib-induced resistance in HCC. Together, these observations indicate that the viability of the drug-tolerant subpopulation requires IGF-1R signaling activation. miR-122 expression is negatively correlated with IGF-1R expression in HCC patients Next, we examined the correlation between expression of miR122 and IGF-1R in 19 HCC sorafenib-resistant patients and 23 HCC sorafenib-sensitive patients. miR-122 was significantly downregulated in the sorafenib-resistant patients compared with the sorafenib-sensitive patients (Fig. 8A). In contrast, IGF-1R was dramatically up-regulated in the sorafenib-resistant patients compared with the sorafenib-sensitive patients (Fig. 8B). These data suggest

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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Fig. 7. Inhibition of IGF-1R activity disrupts tolerance to a variety of cancer drugs in sorafenib-resistant cells. (A) Sorafenib-resistant cells had IGF-1R stably knocked down by lentivirus-mediated delivery of shRNAs directed against IGF-1R. The cells were then cultured with different concentrations of sorafenib for 48 h, and cell viability was measured by CCK-8. (B) In the stable-knockdown cells, colony-formation efficiency was carried out in the presence of sorafenib (5 μM). (C) Huh7- sorafenib-resistant cells were treated by sorafenib (5 μM), PPP (0.01 μM), or NVP-AEW541 (2 μM) alone or the combination of sorafenib with PPP or NVP-AEW541. Colony-formation efficiency was carried out. Drug treatments were repeated every 3 days until colonies were visible. Following treatment, plates were fixed and stained with crystal violet. All experiments were performed in triplicate, and representative stained plates are shown. (D) Parental cells were incubated with sorafenib and IGFII, and colony-formation efficiency was evaluated. (E) Huh7-IGF-1R-OE (IGF-1R-overexpression) cells and Huh7-control cells (1 × 106) were subcutaneously injected into NOD/SCID mice (n = 5), and the tumors were treated with sorafenib gavage (60 mg/kg/day) for 2 weeks. Finally, the tumor volume and tumor weight were analyzed.

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that miR-122 expression was negatively correlated with IGF-1R expression in HCC clinical samples (Fig. 8C and D). These clinical results further indicate that targeting miR-122 and IGF-1R may improve therapeutic benefit to HCC patients receiving sorafenib treatment. Discussion The recent clinical approval of sorafenib has marked as a new era in molecular targeted therapy for advanced HCC [39]. However, an increasingly observed phenomenon in cancer therapy is the socalled re-treatment response [40]. For example, some patients who initially respond well to treatment with sorafenib experience therapy failure later. The relatively rapid acquisition of resistance to cancer drugs remains a key obstacle for successful cancer therapy. The molecular mechanism of sorafenib resistance is still poorly understood. The aim of this work was to examine the cellular alterations and the molecular mechanisms of acquired resistance induced by sorafenib. In our cellular models, miRNA microarray analysis indicated that liver-specific miR-122 was significantly reduced in sorafenib-

resistant cells. These data are consistent with our previous report that miR-122 is involved in modulating the chemoresistance of HCC cells [28]. miR-122 is the most abundant liver-specific miRNA and plays an important role in HCC [25–27]. Sorafenib-resistant and chemo-resistant cells also display down-regulation of the same targets miR-122. These data indicate miR-122 is a predictive tissue specific biomarker for HCC cells’ resistance to sorafenib. We used a miRNA target prediction algorithm to predict miR122 target genes and analyzed the expression of 172 conserved targets in gene expression profiling between parental and sorafenibresistant cells. Based on bioinformatics analysis and our experimental data, IGF-1R is a functional target of miR-122 in HCC cells. miR122 overexpression significantly down-regulated IGF-1R by directly targeting the 3′UTR of IGF-1R mRNA, as shown by luciferase reporter assays. This effect was largely eliminated when the sites in IGF-1R 3′UTR targeted by miR-122 were mutated. These results strongly suggest that miR-122 suppresses the expression of IGF1R by directly interacting with the wild-type 3′UTR of IGF-1R. In addition, the 3′UTR of IGF-1R encompasses a poorly conserved miR-122 cognate site, and we first validated it as target of

Please cite this article in press as: Yanmin Xu, et al., MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.11.034

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Fig. 8. miR-122 expression is negatively correlated with IGF-1R expression in HCC patients. (A, B) Expression of miR-122 (A) and IGF-1R (B) in 19 HCC sorafenib-resistant patients and 23 HCC sorafenib-sensitive patients by RT-PCR analysis. (C, D) miR-122 expression was negatively correlated with IGF-1R expression in the sorafenib-resistant patients (C) and sorafenib-sensitive patients (D).

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miR-122. Both the mRNA and protein of IGF-1R were significantly increased in sorafenib-resistant cells versus parental cells. Altogether, our results show that liver-specific miR-122 helps maintain drug tolerance by regulating its target, the tyrosine kinase receptors IGF-1R. IGF-1R is an important signaling molecule in cancer cells and plays an essential role in the tumorigenesis, anti-apoptosis and resistance to anti-cancer agents [33–35]. The ability of the drugtolerant subpopulation to maintain viability following an otherwise lethal drug exposure appears to involve IGF-1R activation. Furthermore, several published reports describing cell models of acquired resistance to both TKIs and conventional chemotherapy drugs have similarly demonstrated the activation of IGF-1R in drug-resistant subpopulations [29,41,42]. In our established sorafenib-resistant cell models, there was strong evidence that HCC cells rely on IGF-1Rmediated survival pathways to circumvent adverse conditions. Notably, our results suggest that IGF-1R acts by alleviating sorafenibinduced cell apoptosis in drug-tolerant cells, and stable knockdown of IGF-1R expression or inhibition of IGF-1R activity disrupts tolerance to sorafenib. Inhibition of IGF-1R signaling thus appears to be a promising strategy to interfere with the growth and survival of drug-tolerant cells. Although the regulation of IGF-1R activity is poorly understood in sorafenib-induced drug tolerance [43,44], our findings also suggest a crucial role for RAS/RAF/ERK signaling in modifying IGF-1R activity, which involves sorafenib-induced drug resistance. In conclusion, our results indicate that liver-specific miR-122 negatively regulates IGF-1R and that the RAS/RAF/ERK signaling pathway plays a crucial role in sorafenib resistance. We detected abnormal expression of miR-122 and IGF-1R in HCC clinical samples, which

implies a correlation with the emergence of drug resistance. Therefore, combination analysis of miR-122 and IGF-1R could predict to promote drug tolerance, and will provide a rationale for individualized treatment strategies in HCC patients. However, it should be Q6 noted that our results were derived from cell lines that had been removed from their in vivo microenvironment and could not be considered accurate surrogates for clinical tumors. Thus, future studies to assess the roles of miR-122 in vivo and in the clinical context are warranted. Taking these factors into account, we firmly believe that drugs with very selective properties may be employed in combination with liver-specific miR-122 to make HCC treatment more specific and efficient.

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Conflict of interest

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The authors declare no conflicts of interest.

642 Acknowledgements This work was supported by funds from National Natural Science Q7 Foundation of China (No. 81330048 and 81520108025 to C.Q., No. 81301864 to Y.X., No. 81472292 to J.S., and No. 81572464 to J.S.).

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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.11.034.

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