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Introduction

Hepatocellular carcinoma (HCC) has the second highest rate of cancer-associated mortality in men worldwide (1); in addition, chemotherapeutic drug resistance is common in HCC patients (2). Previous studies have indicated that the adjuvant therapies currently available for HCC treatment following surgery are ineffective and inadequate to prevent recurrence (3). Large-scale randomized phase III clinical studies have demonstrated the survival benefits of sorafenib treatment, a unique first-line drug recommended for advanced HCC; however, sorafenib is not commonly used due to its low response rate and drug resistance (4). It is therefore important to elucidate the underlying mechanisms of sorafenib resistance in order to identify potential therapeutic strategies to enhance its efficacy for the treatment of HCC (5–7).

Solid tumors often have a hypoxic internal microenvironment that includes numerous molecular pathways, which provide challenges for therapeutic strategies; this microenvironment is the primary cause of drug resistance to antitumor therapies (8,9). Previous studies have reported that sustained sorafenib treatment may promote hypoxia within tumors, which has been associated with sorafenib-resistance in HCC patients as well as subcutaneous mice models of HCC (10). In addition, it was reported that short-term sorafenib therapy reduced vascularization, resulting in increased tumor hypoxia (11). Therefore, numerous previous studies have aimed to elucidate the potential mechanisms of hypoxia-induced sorafenib resistance mechanisms (5,7,9,10).

Hypoxia-inducible factors (HIFs), which are heterodimers composed of an α and β subunit, have been implicated in the regulation of the hypoxic response (12,13). HIF-1 was reported to mediate the expression of hundreds of genes, including those for vascular endothelial growth factors, glycolytic enzymes and glucose transporters (14,15). In addition, HIF-2 was identified to be involved in implementing the hypoxic response (14,15). HIF-1β was reported to be able to form a heterodimer complex wither either HIF-1α or HIF-2α, both of which undergo oxygen-dependent degradation via the von Hippel-Lindau protein pathway (14,15). In addition, it was reported that targeting HIF-1α may significantly enhance sorafenib antitumor sensitivity under hypoxic conditions (10); however, the association between HIF-2α and sorafenib sensitivity remains to be elucidated.

The present study aimed to investigate the potential association between HIF-2α and sorafenib sensitivity under hypoxic conditions and the mechanisms by which this may proceed.

Materials and methods

Cell culture

Human HepG2, Bel-7402 and Huh-7 HCC cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA); in addition, SMMC-7402 HCC cells were obtained from the Type Culture Collection Cell Bank, Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Shanghai, China), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in 95% air and 5% CO2 or when hypoxic conditions were required, cells were stored at 37°C in a sealed MIC-101 hypoxia chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA) equilibrated with certified gas containing 1% O2, 5% CO2 and 94% N2.

Antibodies (Abs) and reagents

Abs against HIF-2α, β-catenin, C-Myc and proliferating cell nuclear antigen (PCNA), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); Ab against GAPDH was purchased from Beyotime Institute of Biotechnology (Jinan, China); and Abs against Ki-67, HIF-2α small interfering (si)RNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Beijing, China) and sorafenib tosylate was purchased from Bayer Pharmaceuticals Co. (West Haven, CT, USA).

HIF-2α/C-Myc silencing using lentiviral vector-mediated short hairpin (sh)RNA

Silencing of HIF-2α/C-Myc was achieved via lentivival transduction of the following specific shRNA vectors (Santa Cruz Biotechnology, Inc.): HIF-2α-specific shRNA, sc-35316-v; C-Myc-specific shRNA, sc-29226-v; and scramble shRNA control, sc-108080. The process of transduction was executed according to the product protocols. Briefly, HepG2 cells (5×105 cells/well) were seeded in 24-well plates. When the cell were ~50% confluent, the complete medium in the wells was replaced by complete medium with Polybrene (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-134220) at a final concentration of 3.5 µg/ml. In the control shRNA group, 10 µl scramble shRNA (sc-108080) was added to each well and in the HIF-2α shRNA group, 10 µl HIF-2α shRNA (sc-35316-v) was added to each well. The cells were cultured for 8 h, then the mixture of complete medium with Polybrene was replaced with complete medium, 12 h later, the cells were harvested for the following assays.

Cell viability assay

Cell viability was detected using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Beijing, China). In brief, cells (5×103/well) in the control (scramble shRNA-treated group) and the group treated with HIF-2 shRNA were seeded onto 96-well plates and cultured for 12, 24, 48 and 72 h. The culture medium was then replaced with fresh medium containing 10 µl CCK-8 solution. Cells were incubated for a further 2 h at 37°C and the optical density was measured using a PowerWave HT Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm.

Western blot assay

Cells or tumor tissues were homogenized in protein lysate buffer and debris was removed by centrifugation. Protein concentrations in cell/tissue extracts were determined using the BCA Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Inc., Richmond, CA, USA). In brief, equal amounts of protein fractions of lysates were resolved over SDS-PAGE gels (Beyotime Institute of Biotechnology) transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) and immunoblotted as previously described (16,17). The primary antibodies used for western blotting included, monoclonal rabbit anti-human HIF-2α, (1:1,000 dilution, #7096), polyclonal rabbit anti-human-β-catenin (1:1,000, #9562), polyclonal rabbit anti-human c-Myc (1:1,000 dilution, #9402), monoclonal mouse anti-human PCNA (1:2,000 dilution, #2586) and monoclonal mouse anti-human GAPDH (1:2,000 dilution, AG019). The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1,000 dilution, #A0208; Beyotime Institute of Biotechnology) and (HRP)-conjugated goat anti-mouse IgG (1:1,000 dilution, A0216; Beyotime Institute of Biotechnology). Protein bands were developed using ECL reagent (Tiangen Biotech Co., Ltd., Beijing, China) and visualized using the AlphaImager HP System (Naturegene Corporation, Medford, NJ, USA). The protein band intensities were quantified by densitometric analysis using Image J software, version 2.0 (National Institutes of Health, Bethesda, MD, USA).

Immunocytochemistry (ICC)

For ICC, the cells (2×104) were plated on coverslips, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. Subsequently, the sections were incubated with primary anti-β-catenin (1:200 dilution, #9562) overnight at 4°C, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution, #A0208). Subsequently, immunoreactivity was developed with 3,3′-diaminobenzidine (DAB; ZSGB-BIO Biotechnology Co., Ltd., Beijing, China), followed by counterstaining with hematoxylin.

Subcutaneous HCC experiments

All surgical procedures and care administered to the animals were in accordance with institutional ethics guidelines. The study was approved by the ethics committee of Qianfoshan Hospital, Shandong University (Jinan, China). Male BALB/c mice were purchased from the Model Animal Research Center (Nanjing, China). The mice (n=56) were housed in polycarbonate cages (5/cage), provided with food and water ad libitum and maintained on a 12–12 h light-dark cycle at 22±2°C and 55±20% relative humidity. Male BALB/c mice were randomly assigned into four groups (12/group). Eight mice were excluded for different reasons (mortality or no tumor formed). Male BALB/c (5–6 weeks old) mice underwent subcutaneous inoculation in the flank with 1.5×106 HepG2 cells/mice suspended in phosphate-buffered saline (PBS). Sorafenib tosylate was used in vivo experiments. When tumors of ~100 mm3 were detected (~7 days post inoculation), mice were divided at random into the following treatment groups (n=12/group): Control, sorafenib-treated group, HIF-2α siRNA-treated and HIF-2α siRNA + sorafenib-treated. Mice in each group were administered treatment five times/week either intratumorally or orally (p.o). In the sorafenib-treated group, mice were administrated once daily with sorafenib (p.o.; 10 mg/kg; 5 times/week). Equal volumes of 20 nM siRNA and Lipofectamine 2000 were mixed and this solution was further mixed with an equal volume of serum-free medium; 50 µl siRNA transfection solution, containing 250 pmol siRNA, was injected into tumors (5 times/week). Mice in the control group were treated with equal volume PBS (p.o.) and control siRNA (intratumoral injection). The tumor volumes were estimated twice a week according to the formula: π/6xa2xb, where a is the short axis, and b the long axis. All mice were sacrificed by cervical dislocation on day 35.

Quantification of Ki-67 proliferation index

As previously described (18), tumor sections were immunostained with an anti-Ki-67 Ab. Briefly, polyclonal rabbit anti-human Ki-67 (1:50 dilution, SC-15402) was applied overnight at 4°C. After washing, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:75 dilution, #A0208; Beyotime Institute of Biotechnology) for 1 h at 37°C. Immunoreactivity was developed using DAB and counterstained with hematoxylin. Ki-67 positive cells were counted under microscopy with a DM5500B equipped with HCX PL FLVOTAR 5/0.15 and HCX PL FLVOTAR 10/0.15 dry objective lenses (Leica, Solms, Germany) in ten randomly selected high-power fields (magnification, x400). The Ki-67 proliferation index was calculated according to the following formula: Proliferation rate = (number of Ki-67 positive cells/total cell count) × 100%.

Statistical analysis

Values are presented as the mean ± standard deviation and statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed using Chi-Square test or Student's t-test according to the data type. P<0.05 was considered to indicate a statistically significant difference between values.

Results

Targeting HIF-2α enhances the antiproliferation activity of sorafenib under hypoxic conditions

Western blot analysis was used to determine HIF-2α protein expression levels in various HCC cell lines, SNNC-7402, Bel-7402, HepG2 and Huh7, exposed to normoxia or hypoxia for 24 h. All of the cell lines demonstrated low expression levels of HIF-2α under normoxic conditions, while HIF-2α expression was markedly increased under hypoxic conditions in all the selected HCC cell lines (Fig. 1A). Based on this data, these HCC cell lines were further used to detect the inhibitory effects of sorafenib on the proliferation viability of cells under hypoxic conditions. Cells were transfected with HIF-2α shRNA or control shRNA and then treated with sorafenib (10 µM) for 12, 24, 48 and 72 h, following which cell viability was assessed. Compared with the control shRNA, HIF-2α shRNA further enhanced the antiproliferation activity of sorafenib in all of the four cell lines under hypoxic conditions (Fig. 1B).

Figure 1. Targeting HIF-2α enhances the antiproliferation activity of sorafenib under hypoxic conditions. (A) Expression of HIF-2α in hepatocellular carcinoma cell lines SMMC-7402, Bel-7402, HepG2, Huh-7 was detected by western blot analysis following treatment for 24 h under hypoxic conditions. GAPDH served as an internal control. (B) SMMC-7402, Bel-7402, HepG2 and Huh-7 cells were treated with Con shRNA or HIF-2α shRNA in combination with Sorafenib (10 µM) under hypoxic conditions and cell viability was measured following 12, 24, 48 and 72 h of treatment. Values are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.001 vs. Con shRNA. HIF-2α, hypoxia-inducible factor 2α; Con, control; shRNA, short hairpin RNA.

Regulation of cell proliferation-associated protein expression by HIF-2α/C-Myc shRNA

β-catenin is considered to be a key signaling protein in the regulation of cell proliferation (19–21). HepG2 cells were incubated in a sealed hypoxia chamber for 24 h, which resulted in significantly higher levels of β-catenin compared with cells in normoxic conditions (Fig. 2A). Transfection of HIF-2α shRNA markedly reduced the expression of HIF-2α, β-catenin, C-Myc and PCNA in HepG2 cells compared with those transfected with control shRNA (Fig. 2B). As C-Myc is a key downstream protein in β-catenin-mediated cell proliferation (19–21), the effects of C-Myc shRNA transfection were investigated in HepG2 cells. C-Myc shRNA decreased C-Myc and PCNA expression with no effect on the expression of HIF-2α and β-catenin (Fig. 2B). These results indicated that HIF-2α regulated HCC cell proliferation in a β-catenin/C-Myc-dependent pathway under hypoxic conditions.

Figure 2. HIF-2α regulates PCNA expression through β-catenin/C-Myc-dependent pathways under hypoxic conditions. (A) HepG2 cells were transfected with HIF-2α shRNA or Con shRNA, then the expression of β-catenin was detected by immunocytochemistry. (B) HepG2 cells were treated with HIF-2α shRNA, C-Myc shRNA or Con shRNA and cell lysates were analyzed by western blot analysis to detect expression of HIF-2α, β-catenin, C-Myc and PCNA. GAPDH served as an internal control. HIF-2α, hypoxia-inducible factor 2α; PCNA, proliferating cell nuclear antigen; Con, control; shRNA, short hairpin RNA.

Targeting HIF-2α potentiates the antitumor effects of sorafenib in HepG2 xenograft tumor in mice through inhibiting cell proliferation

A gradual increase in xenograft HepG2 tumor growth was observed in the control group compared with the experimental groups. In addition, mice that underwent combination therapy had tumors that were significantly smaller than those treated with sorafenib or HIF-2α siRNA alone (Fig. 3A and B). Furthermore, a reduced number of Ki-67-positive cells were present in tumors treated with either sorafenib or HIF-2α siRNA compared with the control group; while in the combination therapy group, the number of Ki-67-positive cells was significantly reduced compared with that of the independent treatment groups (P<0.01) (Fig. 3C and D). As shown in Fig. 3E, the reduced PCNA expression levels in HIF-2α-transfected cells, as determined by western blot analysis of tumor homogenates, were comparable to the results observed in vitro (Fig. 3E).

Figure 3. Targeting HIF-2α potentiates the antitumor effects of sorafenib in HepG2 xenograft tumor models by inhibiting cell proliferation. HepG2 hepatomas were established. When the tumors reached ~100 mm3 (~7 days post inoculation), they were administered sorafenib, HIF-2α siRNA or a combined treatment (n=12/group). (A) Representative images of tumors from each treatment group. (B) Tumor volume was measured every 7 days for 35 days following treatment. (C) Tumor sections were stained with an anti-Ki-67 antibody in order to detect proliferating cells (magnification, x100). (D) Ki-67 positive cells were quantified in order to calculate the Ki-67 proliferation index. (E) Tumors from mice of each group were homogenized and subjected to western blot analysis in order to detect expression of PCNA. GAPDH served as an internal control. Values are presented as the mean ± standard deviation. *P<0.05 and **P<0.001 vs. control. HIF-2α, hypoxia-inducible factor 2α; siRNA, small interfering RNA; PCNA, proliferating cell nuclear antigen.

Discussion

In the present study it was demonstrated that HIF-2α was involved in sorafenib resistance via the regulation of β-catenin/c-Myc-dependent pathways under hypoxic conditions in HCC. Although sorafenib is first choice of treatment for advanced HCC, it has been reported that this systemic drug has limited benefits and poor response rates (22,23). Solid tumors contain hypoxic cells, which exhibit resistance to antitumor treatments due to an enhanced cellular adaptive response to hypoxia, mediated by HIF-1α and HIF-2α, which promotes survival (14,24–26). HIF-1α synthesis is suppressed following treatment with sorafenib, which results in the altered hypoxic response from HIF-1α to HIF-2α-dependent pathways; this mechanism was reported to result in more aggressive tumor growth (22,23). In the present study, it was demonstrated that targeted HIF-2α knock-down inhibited HCC cell proliferation under hypoxic conditions in vitro; in addition, this acted synergistically with sorafenib to further suppress the growth of HCC tumors in vivo.

β-catenin and C-Myc are important oncogenes, the overexpression of which were reported to be associated with the poorer prognosis of HCC patients (27–30). These oncogenes have numerous biological functions; however, data regarding the role of β-catenin/C-Myc in tumor proliferation are limited in HCC cells. Previous studies have indicated that β-catenin and HIF were frequently co-activated in rapidly growing tumors (31–34). In addition, it was reported that HIF-1α expression was closely associated with Wnt/β-catenin signaling activity (35,36). However, limited studies have investigated the involvement of HIF-2α in β-catenin regulation and cell proliferation (34,37). Furthermore, HIF-2α was demonstrated to interact with β-catenin/T-cell factor (TCF) in order to promote gene transcription (34). Contrary to the results of previous studies (38,39), the present study suggested that HIF-2α may regulate β-catenin/TCF activity through decreasing β-catenin expression under hypoxic conditions in HCC cells; this change may be attributed to different clonal variations.

C-Myc is an important target gene of β-catenin, the expression of which was reported to be significantly upregulated by β-catenin in tumor cells (40–42). In the present study, HIF-2α positively regulated β-catenin/C-Myc expression and C-Myc directly upregulated PCNA expression under hypoxic conditions in HCC cells. However, the exact mechanism of C-Myc-mediated PCNA expression remains to be fully elucidated. Previous studies have indicated that C-Myc may enhance transcription factor accumulation in gene promoter regions, resulting in transcription factor amplification and increased levels of transcripts within the cells gene expression program (43–45). Thus, C-Myc may amplify the output of the PCNA gene expression program in HCC cells under hypoxic conditions.

In conclusion, the present study demonstrated that HIF-2α gene transfer and sorafenib effectively inhibited the proliferation of HCC cells in vivo when administered independently; however, the combination of HIF-2α shRNA and sorafenib treatment resulted in a synergistic effect on inhibiting cell proliferation. The mechanisms underlying these results were indicated to be primarily attributed to the decreased expression of PCNA. Given the distinct transcriptional targets of HIF-2α, the results suggested that downregulating HIF-2α may further enhance the antitumor activity of sorafenib in HCC via β-catenin/C-Myc-dependent pathways.

Acknowledgements

The present study was supported by grants from the National Natural Scientific Foundation of China (nos. 81172331 and 30972890), the Shandong Provincial Science & Technology Development Program, China (no. 2010GSF10230) and the Youth Foundation of CSCO-Bayer Schering Pharma for Hepatocellular Carcinoma (no. Y-B2011-012).

References