Hypoxia-inducible factor-1 up-regulates the expression of Toll-like receptor 4 in pancreatic cancer cells under hypoxic conditions

Pancreatology 12 (2012) 170e178

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Original article

Hypoxia-inducible factor-1 up-regulates the expression of Toll-like receptor 4 in pancreatic cancer cells under hypoxic conditions Ping Fan a, Jian-Jun Zhang a, Bo Wang a, Han-Qing Wu a, Si-Xing Zhou a, Chun-You Wang a, Jing-Hui Zhang b, Yuan Tian b, He-Shui Wu a, * a b

Department of Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1277, Jiefang Road, Wuhan, Hubei 430022, China Laboratory of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

a b s t r a c t Keywords: Toll like receptor 4 Pancreatic cancer Panc1 cells Hypoxia-inducible factor 1

Background & aims: Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that Toll-like receptor 4 (TLR4) plays a significant role in cancer invasion and progression. In this study, the correlation between the expression of TLR4 and the change of the protein level of Hypoxia-inducible factor-1 alpha (HIF-1a) was studied. Methods: We examined 84 human pancreatic cancer tissues for expression of HIF-1a and TLR4 proteins. Panc-1 cells were exposed to normoxia (20% O2) or hypoxia (<1% O2) or treated with CoCl2. TLR4 protein was analyzed by flow cytometry and immunostaining. Growth studies were conducted on cells with the HIF-1a inhibition isolated from stable transfected cell lines. Finally, TLR4 protein was detected by immunohistochemistry in vivo tumors. Results: There was a positive correlation between TLR4 and HIF-1a protein in pancreatic cancer tissues. Hypoxic stress induced TLR4 mRNA and protein expression in Panc-1 cells. Cells transfected with HIF-1a siRNA showed attenuation of hypoxia stress-induced TLR4 expression. In vivo growth decreased in response to TLR4 and HIF-1a inhibiton. Transient HIF-1a siRNA treatment could effectively curb tumor growth in vivo. Conclusion: These results suggest that TLR4 expression in pancreatic cancer cells is up-regulated via HIF1a in response to hypoxic stress and underscore the crucial role of HIF-1a-induced TLR4 in tumor growth. Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

1. Introduction Pancreatic cancer is one of the most lethal solid tumors of the gastrointestinal tract. These tumors are highly invasive and show extensive metastatic potential [1]. Although the molecular mechanisms linking genetic changes to the aggressive nature of this disease are poorly understood, many growth factors and their receptors are overexpressed in human pancreatic cancer [2,3]. Toll-like receptors (TLRs) are a family of pattern recognition receptors that are best-known for their role in defending their host from infection [4]. Mounting evidence also suggests that TLRs have an important role in maintaining tissue homeostasis by regulating the inflammatory and tissue repair responses to injury [5]. To date, at least 13 types of human TLRs, with different ligand specificities,

* Corresponding author. Tel./fax: þ86 27 85351621. E-mail address: [email protected] (H.-S. Wu).

have been identified. Our understanding of the role of TLRs in cancer is extremely limited, however, some studies have suggested that TLRs may have a universal role in cancer [6e8]. There is a positive association between TLR4 expression and hypoxic disease, and its expression is upregulated in various tissues of patients with ischaemiaereperfusion injury, including brain, lung, heart and kidney [9e13]. Moreover, up-regulated expression of TLR4 has been detected in many tumor cell lines or tumors, such as breast cancer cell, gastric carcinoma cell [14], ovarian cancer cell [15] and prostate cancer cell [16]. Kelly et a1 [15] found that activation of TLR4 signaling promotes growth and chemoresistance of epithelial ovarian cancer cells. Conversely, inhibition of TLR4 signaling delays tumor growth and prolongs the survival of animals [17,18]. Hypoxia is a common feature of solid tumors. HIF-1a is a major survival factor for tumor cells growing in a low oxygen environment. Moreover, it plays a critical role in inducing hypoxia-related gene expression and cellular responses [19]. HIF-1 is a heterodimer

1424-3903/$ e see front matter Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved. doi:10.1016/j.pan.2012.02.015

P. Fan et al. / Pancreatology 12 (2012) 170e178

composed of an inducible HIF-1a subunit and an HIF-1b subunit. While HIF-1b is constitutively-expressed, HIF-1a is tightly regulated by oxygen. Therefore, the overall activity of HIF-1 is regulated by the intracellular level of HIF-1a. HIF-1a protein degradation is regulated by O2-dependent prolyl hydroxylation, which targets the protein for ubiquitylation by E3 ubiquitin-protein ligases. These ligases include the von HippeleLindau tumor-suppressor protein (VHL), which binds specifically to hydroxylated HIF-1a. Ubiquitylated HIF-1a is rapidly degraded by the proteasome at normoxia [20]. HIF-1a synthesis leads to dimerization with HIF-1b, which results in the stabilization and translocation of HIF-1a into the nucleus to form the active HIF-1 transcription factor [21]. Indeed, recent studies have confirmed that HIF-1a over-expression is associated with a poor prognosis in pancreatic cancer patients. The TLR4 ligand lipopolysaccharides (LPS) induces HIF-1a expression in murine macrophages [22], in dendritic cells (DCs) and human myeloid monocytic leukaemia cells [23], which suggests that complex cross-talk between the HIF-1a and TLR4 may exist. We hypothesized that TLR4 expression may be upregulated by hypoxic conditions to facilitate pancreatic tumor growth. In this case, regulation of HIF-1a-induced TLR4 expression might represent a novel therapeutic target in pancreatic cancer.

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2.2. Cell culture and hypoxic treatment The panc-1 cell line was obtained from the ATCC (American Type Culture Collection) and cultured in Dulbecco’s Modified Eagle Medium with GlutaMAXÔ (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin at 37  C in a humidified 5% CO2 atmosphere. To expose cells to a hypoxic environment, cells were either placed in an airtight chamber with inflow and outflow valves infused with a gas mixture (5% CO2, 3% H2, 92% N2) that maintained a low oxygen tension (<1%) or treated with 100 mM CoCl2, which is been widely used to induce hypoxic stress.

2.3. Reagents and antibodies The hypoxia surrogate CoCl2 was purchased from the SigmaeAldrich. The full length HIF-1a expression vector (HAeHIFe1alpha-pcDNA3) was purchased from www.addgene. com. Luciferase reporter plasmids containing the TLR4 promoter were obtained from Qiagen. The mouse antieHIFe1a monoclonal antibody and the rabbit anti-TLR4 (sc-10741) antibody were obtained from Santa Cruz Biotechnology Inc., (CA, USA).

2. Material and methods 2.1. Patients and tissues

2.4. Immunohistochemical staining and evaluation

The study group consisted of 84 patients with pancreatic cancer who underwent surgery at the Department of Pancreatic Surgery of Union Hospital, HUST. (Wuhan, China) between December 2007 and April 2010. The anonymous use of redundant tissue for research purposes is part of the standard treatment agreement with patients in our hospital. The median age of the patients was 53 years, with a range of 25e76 years. None of the patients received preoperative radiotherapy or chemotherapy. Fresh tissue samples were obtained from all of the resected specimens and were rapidly frozen at 80  C for storage until analysis. Three of the patients, who had distinct pancreatic tumors, showed distant metastasis. After pancreatectomy, almost all of the patients received postoperative systemic chemotherapy. The tumor stage was defined by the sixth edition of the classification tumor, nodes, metastasisclassification (TNM) of the International Union Against Cancer (UICC). Table 1 shows an association between TLR4 staining pattern, expression of HIF-1a and clinicopathological features. This information was re-assessed independently by two investigators.

Immunohistochemical staining was performed using Histostain-Plus Kits from Zymed Laboratories, Inc. (San Francisco, CA, USA). The antibodies mouse anti-human HIF-1a monoclonal antibody and rabbit anti-human TLR4 antibody were diluted as described as recommended by the manufacturer. Normal mouse IgG was used instead of primary antibody as a negative control. To compare the regional expression of HIF-1a and TLR4 in human pancreatic cancer, the expression of both proteins was evaluated in serial sections of selected cases that showed both HIF-1a and TLR4 expression. Immunoreactivity images were obtained at a 400  magnifications using a microscope and computer system, and two independent investigators (Ping Fan and Bo Wang) calculated the number of positive cells in a minimum of 10 fields per specimen. When an evaluation differed, the final decision was made by a consensus agreement. The intensity of staining was classified by the percentages of the cancer cells stained and the intensity of the staining, as described previously [24].

2.5. Reverse transcriptionepolymerase chain reaction (RT-PCR) analysis Table 1 Associations between TLR4 staining pattern, expression of HIF-1a and clinicopathological features. TLR4 status Pathological stage

Tumor status

Necrosis Nodal status HIF-1a

I II III IVA IVB T1 T2 T3 T4 Yes No N0 N1 Positive Negative

þve

ve

15 16 22 6 5 4 14 33 13 43 21 45 19 59 5

7 6 4 2 1 9 5 5 1 7 13 12 8 7 13

P-value 0.405

<0.001

0.010 0.390 <0.001

Total cellular RNA was purified from using TRIzol (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized, and equal amounts of cDNA were subjected to real-time PCR, which was performed using the Quantitect Reverse Transcription Kit (Qiagen, Valencia, CA, USA) and an Mx3000PÔ real-time PCR detection machine (Stratagene, La Jolla, CA, USA).The primer sequences for the TLR4 gene were as follows: 50 -ACCTGTCCCTGAACCCTATGAA-30 (forward) and 50 -CTTCTAAACCAGCCAGACCTTG-30 (reverse); the primer sequences for the HIF-1a gene were:50 -TGAAGTGTACCCTAACTAGCCG- 30 (forward) and 50 AATCAGCACCAAGCAGGTCATAG- 30 (reverse); and the primer sequences for b-actin were:50 -GAAACTACCTTCAACTCCATC-30 (forward) and 50 -CGAGGCCAGGATGGAGCCGCC-30 (reverse).The specificity of the amplified PCR products was assessed by a melting curve analysis. The fold change in gene expression was calculated as previously described [25].

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2.6. Western blot Total proteins were extracted from Panc-1 cells. HIF-1a monoclonal antibody was purchased from BD Biosciences. b-Actin polyclonal antibody was purchased from Santa Cruz. Total protein concentrations were determined using a BCA protein assay kit (PIERCE) and by Biophotometer (Eppendorf, Germany). Total proteins were separated on 9% SDS-PAGE gel for HIF-1a and 11% for b-actin, transferred to polyvinylidene difluoride membranes by Mini Trans-Blot (Bio-Rad). The blot membrane was then incubated with primary and secondary antibodies and treated with enhanced chemiluminescence detection reagents (Amersham, UK). The specific blotting band was recorded on film. The results were analyzed by ImageJ software. 2.7. Flow cytometric analysis Panc-1 cells were labeled with a mouse phycoerythrin (PE)conjugated anti-human TLR4 antibody and a mouse PE-conjugated isotype immunoglobulin G2a (IgG2a) antibody (as an isotype control) (BioLegend, San Diego, CA, USA). All staining procedures were performed at 4  C in Pharmingen Stain Buffer (BD Pharmingen, San Diego, CA, USA). Cells were analyzed by flow cytometry using an EPICS XL-MCL (Beckman Coulter, Fullerton, CA). All experiments were repeated six times. 2.8. Immunofluorescence Panc-1 cells were grown on glass 18-mm diameter coverslips (Fisher Scientific, Pittsburgh, PA). The cells were labeled with perchlorate (DiO) VybrantÔ30 ,30 -dioctadecyloxacarbocyanine (Molecular Probes Inc., Eugene, OR), fixed with 4% PFA, washed three times with phosphate- buffered saline(PBS), and blocked with PBS 1  containing 2% BSA and 10% FBS for 30 min. The cells were subsequently incubated with an anti-TLR4 antibody in blocking buffer overnight at 4  C followed by incubation with fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody (SigmaeAldrich). Cells were washed with PBS and costained with DAPI. The slides were examined with an FV 1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) equipped with 200  objectives. The images were obtained with FLUOVIEW software (Olympus Corporation, Tokyo, Japan). 2.9. Transfection and luciferase assay Panc-1 cells were transfected with a luciferase plasmid and an HIF-1a expression plasmid using the SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. A heat-shock protein b-galactosidase plasmid was cotransfected as an internal control. The total numbers of transfected plasmids were kept consistent by supplementing with the

corresponding empty vector. The enzymatic activity of luciferase and b-galactosidase was determined with the Luciferase Assay System and the b-galactosidase Enzyme System (Promega). Luciferase activity was normalized against b-galactosidase activity. 2.10. RNA interference and cell transfections Human-specific HIF-1a small interfering (siRNA) and a nontargeting control siRNA were synthesized and purified by Qiagen. The HIF-1a siRNA targeted nucleotides 1663e1681 of the HIF-1a mRNA sequence (NM001530.3) and comprised of sense 50 CUGAUGACCAGCAACUUGAdTdT-30 . The inverted HIF-1a1 control did not target any gene and comprised of sense 50 -AGUUCAACGACCAGUAGUCdTdT-30 . Panc-1 cells were plated at a concentration of 2  105 cells per well in six-well plates. The following day, cells were transfected with siRNA using the HiperFect transfection reagent (Qiagen, Valencia CA) for 6 h, and cells were subsequently placed in fresh medium under normoxia or hypoxia for 12 h. 2.11. Creation of short hairpin RNA (shRNA) stable transfections The pSilencer 2.1 U6-hygro plasmid from Ambion was used to express an shRNA controlled via the U6 promoter according to the manufacturer’s protocol. The shRNA 1589 sequence was used to create pSil2.1_hygro-1589, and the negative control, pSil2.1_hygroNeg. Panc-1 cells were transfected with the Fugene transfection reagent (Roche, Alameda, CA), and transfectants were selected with 300 ug/ml hygromycin in Dulbecco’s Modified Eagle Medium. 2.12. Xenograft of panc-1 cells in nude mice and immunohistochemistry to assess the expression of TLR4 in xenograft tumor The stably transfected Panc-1 cell line and control cells were injected s.c. in athymic nude mice. Each aliquot of approximately 2  106 cells suspended in 100 ml of PBS containing 20% of Matrigel Growth Factor Reduced (Becton Dickinson Labware, Flanklin, NJ, USA) were injected subcutaneously into the flank of 8-week-old male BALB/c nude mice. For each group, 12 animals were used. Tumors were allowed to establish for 2 weeks and then their size was measured with calipers twice weekly for up to 8 weeks. After 8 weeks, all mice were sacrificed and the tumors were dissected and rapidly frozen at 80  C for storage until analysis. Tumor volume measurements were made by a single blinded observer to prevent observer bias and to avoid interpersonal differences in caliper tumor measurement. Paraffin-embedded tumor tissues were cut at 4 mm serial sections and processed as usual. The sections of all specimens were stained with hematoxylin and eosin (H&E) routinely. Immunohistochemistry was carried out in accordance with the manufacturer’s instructions. of streptavidin-peroxidase

Fig. 1. Correlation of TLR4 and HIF-1a expressions in pancreatic cancer tissues. (A) Brown nuclear staining with HIF-1a is found in tumor cells. (B) Staining with TLR4 is predominantly membranous (magnification  400). HIF-1a expression co-localizes with TLR4 expression in consecutive sections of same tumor tissue, suggesting a coincident spatial expression. (C) Correlations between the percentage of TLR4 and HIF-1a-positive cells. A robust correlation was observed (r ¼ 0.623, P < 0.001).

P. Fan et al. / Pancreatology 12 (2012) 170e178

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Fig. 2. Hypoxic stress induces TLR4 expression in pancreatic cancer cells. Cells were incubated under normoxia (20% O2) or hypoxia (<1% O2) or treated with CoCl2 (100 mM) for (a) the indicated times. The mRNA levels of TLR4 at different time-points were analyzed by RT-PCR and normalized with b-actin expression (meanþ/e SEM were shown in Table 2). (b) Surface protein expression of TLR4 was analyzed by flow cytometry after cells were stained with phycoerythrin (PE)-conjugated anti-TLR4 antibody (TLR4 Ab) or isotype control antibody (immunoglobulin G2a; IgG2a). (c) TLR4 protein expression was analyzed by immunostaining and confocal microscopy using an anti-TLR4 antibody together with fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody and a fluorescent membrane marker (DiO: 30 ,30 -dioctadecyloxacarbocyanine perchlorate). Values are means  SEM (n ¼ 3e5). Veh, vehicle (n ¼ 4e5).

(SP) staining kit and diaminobenzidine (DAB) kit were supplied by Maixin-Bio, Fuzhou, China. Digital images of positively stained fields were assessed by measuring the optical density (OD) of stained regions of tumor tissue. Brown granules in the cytoplasm or/and on the cell membrane represented positive staining, and

staining intensity indicated the expression level of TLR4. Photographs were taken at high magnification (400  ) for analysis. The expression of TLR4 was analyzed using the Image Pro-Plus software (ver. 6.0; Media Cybernetics, Silver Spring, MD), according to the method developed by Xavier et al. [26].

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Table 2 The mRNA levels of TLR4 at different time-points. Hours

3h 6h 12 h

TLR4 mRNA fold induction (mean  SEM)

TLR4 mRNA fold induction (mean  SEM)

20%O2

<1%O2

P

Veh

CoCl2

P

1.550  0.156 1.556  0.693 1.460  0.381

2.148  0.437 3.690  1.392 4.186  1.544

0.021 0.015 0.004

1.370  0.389 1.814  0.182 1.928  0.329

2.624  1.038 4.074  2.042 4.486  1.715

0.035 0.039 0.011

2.13. SiRNA mouse treatment siRNAs with O-methyl (OMe)-modified nucleotides were used for in vivo experiments using this pattern (sense 50 eOMeXeOMeXeOMeXeOMeXeOMeXeXXXXXXXXeOMeXe OMeXeOMeXeOMeXeOMeXed(TT)e30 ) as previously described [27]. The siRNA sequences used in this study were HIF-1a siRNA (sense 50 -CUGAUGACCAGCAACUUGAdTdTd(TT)-30 ) and nonsilencing(control) siRNA (sense 50 -AGUUCAACGACCAGUA0 GUCdTdTd(TT)-3 ). Cells were seeded in 12-well plates (4  104 cells per cm2) and incubated for 24 h. OMeesiRNAs were transfected at a concentration of 45 nM using the JetPEI Transfection Reagent (Polyplus). Non-silencing unmodified or OMeesiRNA was used as negative control. For in vivo experiments, institutional guidelines for the research animals were followed. Sixweek-old male nude mice were anesthetized by intra peritoneal (i.p.) injection of a ketamine-xylazine mix (100 mg/kge10 mg/kg), and 1  107 (control OMeesiRNA or HIF-1a OMeesiRNA) Panc-1transfected cells were subcutaneously injected with 0.1 ml of Matrigel to develop xenografts. One day after injections, 30 mg of OMeesiRNAs and 25 mL of JetPEI 4  were incubated in 160 mL of JetPEI buffer (10 mM HEPESe150 mM NaCl, pH 7.4) for 1 h and i.p. injected every third day for 8 weeks. Tumor volume measurements were performed once weekly and calculated by the formula

length  width  depth  0.5236 as previously described [28]. Each experimental group consisted of 6 mice and the experiment was repeated twice.

3. Results 3.1. Correlation of TLR4 and HIF-1a expressions in pancreatic cancer tissues The expression and localization of TLR4 and HIF-1a in 84 resected pancreatic cancer tissues was determined by immunohistochemistry. HIF-1a expression was found in 66/84 (79%) of pancreatic cancer samples, and TLR4 expression was found in 64/84 (76%) cases. The 64 cases with TLR4 expression showed positive coexpression of HIF-1a in 59 cases. Furthermore, we showed that the areas of the serial sections that expressed TLR4 overlapped with the areas that expressed HIF-1a (Fig. 1A,B). In a bivariate analysis, there was a strong correlation between the percentage of TLR4 positive cells and the percentage of HIF-1a positive cells (r ¼ 0.623, P ¼ 0.000 < 0.001) (Fig. 1C). TLR4 staining was also observed in 59/ 66 (89%) of the tumors with HIF-1a expression. The presence of TLR4 expression was significantly associated with necrosis (Table 1) (c2-test, P ¼ 0.010).

Fig. 3. Effects of HIF-1a siRNA on HIF-1a expression under hypoxia in Panc-1 cells. Panc-1 cells transfected with either siRNA targeting HIF-1a or control siRNA were exposed to normoxia (20% O2) or hypoxia (<1% O2) or treated with CoCl2 (100 mM) for 12 h. mRNA levels of HIF-1a were determined by RT-PCR. Expression of HIF-1a protein was determined by Western blotting. (a)Real-time PCR shows that siRNA targeting HIF-1a significantly inhibited the expression of HIF-1a mRNA in Panc-1 cells as compared cells treated with control siRNA (n ¼ 5).(b)Western blot showed that siRNA targeting HIF-1a sigfnificantly suppressed expression of HIF-1a protein after 12 h under hypoxia but the control siRNA group was uneffected. Means  SEM (n ¼ 5). Veh ¼ vehicle.

P. Fan et al. / Pancreatology 12 (2012) 170e178

3.2. Hypoxic stress induces TLR4 expression in pancreatic cancer cells The messenger RNA (mRNA) levels of TLR4 were determined by RT-PCR when cells were exposed to hypoxia or treated with CoCl2 for 3, 6 and 12 h. Both hypoxia and CoCl2 enhanced the levels of TLR4 mRNA in a time-dependent manner (Fig.2a,Table 2). Protein levels of TLR4 were markedly increased after exposure to hypoxia or CoCl2 for 12 h (P ¼ 0.000 and 0.004, respectively Fig.2b,c). 3.3. HIF-1a is involved in the hypoxia-enhanced the expression of TLR4 in vitro To characterize the hypoxia-induced TLR4 expression, we tested the role of HIF-1, using HIF-1 siRNA. The siRNA targeting HIF-1a reduced the level of HIF-1a mRNA (P ¼ 0.015, P ¼ 0.008 respectively Fig. 3a) and the expression of HIF-1a protein (P ¼ 0.009 Fig. 3b). Up-

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regulation of TLR4 mRNA, induced by hypoxia or CoCl2, was significantly attenuated when HIF-1a expression was knocked down by siRNA (P ¼ 0.004 and 0.001 respectively Fig.4a). Consistent with this observation, the HIF-1a siRNA also decreased TLR4 protein expression in response to CoCl2 (P < 0.001, Fig. 4b). Furthermore, to determine whether HIF-1a is involved in TLR4 expression, we employed a gain-of-function approach and overexpressed HIF-1a using an HIF-1a plasmid. Transfection of the HIF-1a expression plasmid resulted in an increase in the level of nuclear translocated HIF-1a protein in cells under hypoxic conditions compared with mock-transfected cells under hypoxic conditions (data not shown). Over-expression of HIF-1a increased expression of the luciferase reporter that was driven by the TLR4 promoter and the TLR4 mRNA levels in Panc-1 cells treated with CoCl2 (P ¼ 0.006 and 0.009, respectively Fig. 4c,d). In contrast, HIF1a over-expression in Panc-1 cells that were not exposed to CoCl2 did not demonstrate enhanced reporter expression or TLR4

Fig. 4. HIF-1a is involved in hypoxia-enhanced the expression of TLR4. Panc-1 cells transfected with either siRNA targeting HIF-1a or control siRNA were exposed to normoxia (20% O2) or hypoxia (<1% O2) or treated with CoCl2 (100 mm) for 12 h (a) TLR4 messenger RNA levels were determined by RT-PCR. (b) TLR4 expression was analyzed by flow cytometry. (c) Panc-1 cells were cotransfected with a luciferase construct driven by the TLR4 promoter and an expression plasmid for HIF-1a, and the cells were treated with CoCl2 (100 mm) for 24 h. Cell lysates were analyzed for luciferase activities. The relative luciferase activity (RLA) was calculated after normalization against b-galactosidase activity. (d) Panc-1 cells were transfected with the HIF-1a expression plasmid and treated with CoCl2 (100 mm) for 8 h. TLR4 mRNA levels were determined by RT-PCR. Values are means  SEM (n ¼ 3e4). Veh, vehicle (n ¼ 4e5).

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transcript levels, which was likely a result of the rapid degradation of HIF-1a under normoxic conditions (P ¼ 0.325 and 0.456 respectively Fig. 4c,d). 3.4. Stable knockdown of HIF-1a significantly reduces tumor volume and the expression of TLR4 in vivo In vivo growth studies were performed using Panc-1 cells that had been transfected with either HIF-1 shRNA or control shRNA and grown on the flanks of nude mice. The mice were monitored for 8 weeks before they were sacrificed. From the graph of tumor growth curve, we found that there was statistical difference in tumor growth between the control group and the HIF-1 shRNA group after 30 days (P ¼ 0.0075, Fig. 5a). Moreover, it was interesting that the growth of xenograft tumor had no difference between the control group and the HIF-1 shRNA group in the early stage such as the fifth day, the eighth day, the 21st day and the 24th day. From immunohistochemistry, we found that there was significant difference between the control group and the HIF-1 shRNA group about in the expression of TLR4 in pancreatic xenograft tumor (P ¼ 0.0276, Fig. 5b,c).

3.5. Transient HIF-1a siRNA treatment reduces tumor volume Native siRNAs are rapidly degraded in vivo, and mammalian cells do not readily take up naked nucleic acids. Therefore, we chose chemically modified siRNAs in which the 5 first bases at the 30 and 50 ends were 20 -O-methylated. These modifications increase siRNA resistance to nucleases while maintaining their activity [27]. We used Jet-PEI, a polyethylenimine mixture of undisclosed composition, to target the siRNAs to the pancreatic cancer-derived Panc-1 cells. Jet-PEI facilitates extremely efficient transfection in vivo with low toxicity [29,30]. This strategy was validated in vitro before being employed in in vivo experiments. Two days after transfection of Panc-1 cells, TLR4 mRNA expression was assayed by RT-PCR and HIF-1a mRNA expression was inhibited by more than 80% was observed (data not shown). Nude mice were subcutaneously transfected with 1  107 Panc-1 cells that had received either control OMeesiRNA or HIF-1a OMeesiRNA and the development of xenografts was monitored. In addition, mice were treated with control OMeesiRNA or HIF-1a OMeesiRNA that was administered in a complex with JetPEI. The siRNA injection was repeated every third day. The tumor size was measured weekly for eight weeks

Fig. 5. Stable knockdown of HIF-1a significantly reduces tumor volume and the expression of TLR4 in vivo. BALB/c nude mice were injected subcutaneously in the flank with Panc-1 cells that had been transfected with either HIF-1a shRNA or control shRNA. After 8 weeks, formalin-fixed primary tumor sections from each mouse were prepared and stained with an anti-TLR4 antibody. The brown color denotes TLR4 in primary tumors. (a) Graph showing mouse flank (panc-1 cells) tumor volume as measured by digital calipers. The tumor growth in experimental group was significantly slower than that in the control groups (P < 0.001, n ¼ 12 per group; both groups are verified by repeated analysis by ANOVA and Tukey’s multiple comparison test, P < 0.01). (b) Immunohistochemical staining for TLR4 with monoclonal antibody-TLR4(1:200 dilution).The brown signals represent positive staining for TLR4. 1, HIF-1a shRNA xenograft tumor (n ¼ 18).; 2, Control shRNA xenograft tumor(n ¼ 16). (Magnification,  400) (c) The positivity rates of TLR4 were calculated and illustrated as shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Fan et al. / Pancreatology 12 (2012) 170e178

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Fig. 6. Transient HIF-1a siRNA treatment reduces tumor volume in vivo. Panc-1 cells were seeded onto 12-well plates (5  104 cells per cm2) and transfected with control siRNA (non-silencing) or HIF-1a OMe siRNAs at a concentration of 45 nM in JetPEI Transfection Reagent. After 24 h, nude mice were injected subcutaneously with 1  107 Panc-1 transfected cells with 0.1 ml of Matrigel to facilitate xenografts development. One day after injections, 20 mg of OMeesiRNAs were injected intraperitoneally as described in the experimental procedures and the injection was repeated every 3 days for 8 weeks (Fig. 5). Tumor volume was measured weekly. Values are expressed as the mean of n ¼ 6  S.D. of two independent experiments.

following injection of the cells. Panc-1 cells that were transfected with control OMeesiRNA produced tumors in all athymic nude mice (6/6) whereas HIF-1a-deficient Panc1 cells did not (0/6) (Fig. 6). 4. Discussion Recent evidence suggests that TLRs play an important role in maintaining tissue homeostasis by regulating the inflammatory and tissue repair responses to injury. Specifically, TLR4 is an important member of the TLR family and is present in tumors, such as ovarian cancer, prostate cancer and colorectal cancer. The expression of TLR4 in tumor cells plays a role in immune surveillance and facilitating tumor growth resistance and chemoresistance [31]. Moreover, it has been reported that reduced expression of TLR4 resulted in a dramatic reduction of cancer cell viability [32]. However, TLR4 expression is not static but rather is modulated dynamically in response to environmental stresses such as hypoxic stress. It is not yet clear if hypoxic stress induces TLR4 in pancreatic cancer cells. In this study, we have demonstrated that expression of TLR4 was upregulated in Panc-1 cells that were exposed to hypoxic stress. Up-regulation of TLR4 mRNA in Panc-1 cells was observed after exposure to 3 or 6 h of hypoxic stress. Kuhlickeet et al. [33] showed that protein expression of TLR2 and TLR6 was induced by relatively long-term exposure to hypoxic conditions for 24e72 h in human microvascular endothelial cells. However, the TLR4 mRNA level was not changed by treating bone-marrowderived dendritic cells with hypoxia for 24 h. Although differences in the duration of hypoxia and the types of cells may account for differential cellular responses in TLR expression, these results suggest that expression of TLRs such as TLR4 and TLR2 can be dynamically regulated by hypoxic stress. TLR4 initiates signals through the sequential recruitment of myeloid differentiation protein 88(MyD88), which activates downstream mediators, such as NF-kB, to activate genes that encode proinflammatory cytokines. The MyD88 signaling pathway downstream of TLR4 induces hepatocarcinogenesis and intestinal tumorigenesis [34,35]. NF-kB is a multifunctional transcription factor that affects tumor growth and metastasis [36]. Ikebe et al. [37] showed that TLR4/MyD88/NFkB signaling pathway plays a significant role in connecting inflammation to cancer invasion and progression. Utilizing in vitro systems, researchers have reported that HIF-1a activates NF-kB

[36] and that NF-kB controls HIF-1a transcription [38] Lee et al. [39] demonstrated that TLR4 signaling influenced tumor growth and, that TLR4 signaling was a critical upstream activator of NF-kB and that over-expression of heat shock proteins (HSPs) in tumor cells increased tumor growth and inflammation. Spirig et al. [40] reported that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induced the expression of HIF-1a in human monocyte-derived dendritic cells in a time-dependent manner. These studies are consistent with a role for the TLR4 signal pathway in regulating HIF-1a transcription. Here, we found that the hypoxia-induced TLR4 expression was HIF-1a dependent demonstrated by the ability of HIF-1a siRNA to inhibit hypoxiainduced TLR4 upregulation and the ability of HIF-1a expression plasmid to enhance TLR4 expression in hypoxia condition in Panc1 cells. These results suggest that HIF-1a promotes TLR4 expression under hypoxic conditions that maintain the level of HIF-1a protein is maintained. According to an analysis of the human Tlr4 gene (UCSC Genome Browser, accession no. uc004bjz1), the human TLR4 promoter contains at least two putative HREs located at 2811 to 2814 and 1185 to 1188. However, it has not been entirely clear which putative HREs are involved in the regulation of TLR4 expression under hypoxia. Kim et al. [41] reported that HIF-1a binds to mouse TLR4 promoter region at the hypoxic putative hypoxia-responsive element (pHRE) (50 -CGTG-30 ) located at 407 to 404. Moreover, deletion of the CGTG region, or mutation of the CGTG to AAAG, led to a significant reduction of TLR4 promoter reporter activity in response to HIF-1a overexpression, demonstrating that this site was important for HIF1-mediated TLR4 expression. Together, these results suggested that TLR4 induction by hypoxia was determined, at least in part, by HIF-1. Our HIF-1a RNAi knock down results indicate that the protein is a required for sustained pancreatic tumor growth. The replication of siRNAs indicates that this response is produced by an off-target effect. We show that an injected siRNA, complexed to JetPEI, is an effective silencer of HIF-1a expression and inhibitor of the tumor growth in vivo. This mode of delivery has direct clinical application because the treatment is transient and does not involve the use of viral vectors or other permanent DNA modifying procedures. Moreover, our shRNA results also indicate that the reduction of HIF1a had an obvious effect on reducing the expression of TLR4 of Panc-1 cells in vivo. This result further supports a role for HIF-1a in mediating TLR4 expression in pancreatic cancer.

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