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. 2017 Jun 27;8(26):42887-42900.
doi: 10.18632/oncotarget.17157.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

Nanae Harashima  1 , Keizo Takenaga  2 , Miho Akimoto  2 , Mamoru Harada  1
Affiliations

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

Nanae Harashima et al. Oncotarget. .

Abstract

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy.

Keywords: HIF-2α; TRAIL; hypoxia; pancreatic cancer; surviving.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no competing interest.

Figures

Figure 1
Figure 1. Transfection of HIF-2α siRNA increases TRAIL sensitivity of pancreatic cancer cells
(a) Panc-1 and AsPC-1 cells were transfected with the indicated siRNAs and cultured under hypoxic (1% O2) or normoxic conditions for 24 h, and then subjected to immunoblotting using nuclear extracts. E2F-1 was used as a loading control. N: normoxic, H: hypoxic. (b) siRNA-transfected cancer cells were cultured with TRAIL under normoxic or hypoxic conditions for 48 h. Cell viability was analyzed using the WST-8 assay. Similar results were obtained from two independent experiments. *p < 0.05, **p < 0.01 were compared to negative control siRNA. (c) Two cell lines were incubated with TRAIL (100 ng/mL, 24 h for Panc-1 or 50 ng/mL, 12 h for AsPC-1) under normoxic conditions and flow cytometric analysis was performed. Similar results were obtained from two independent experiments. (d) The results are presented as means ± SD from triplicate experiments. The open and closed bars represent the data of control and TRAILtreatment, respectively. ** p < 0.01.
Figure 2
Figure 2. Caspase-dependent apoptosis of HIF-2α siRNA-transfected and TRAIL-treated Panc-1 cells
(a) siRNA transfected Panc-1 cells were treated with various doses of TRAIL for 6 h. Protein lysates from whole cells were assayed using immunoblotting. α-tubulin was used as a loading control. (b) siRNA transfected Panc-1 cells were cultured with TRAIL (100 ng/mL) in the presence of the indicated caspase inhibitors (20 μM). The cells were examined using flow cytometric analysis. The number represents the percentages of each subset. zVAD, pan-caspase inhibitor (z-VAD-FMK); C8i, caspase-8 inhibitor (z-IETD-FMK); C9i, caspase-9 inhibitor (z-LEHD-FMK). Similar results were obtained from two independent experiments. (c) The results of Annexin V+ cells (%) are presented as means ± SD from triplicate experiments. **p < 0.01. (d) siRNA transfected Panc-1 cells were treated with the indicated concentration of TRAIL for 6 h. The protein expression levels of c-FLIP were determined using immunoblotting. α-tubulin was used as a loading control. (e) Similarly, the lysates were used for immunoblotting to examine the expression of the indicated proteins. β-actin was used as a loading control.
Figure 3
Figure 3. Effect of knockdown of either HIF-1α or HIF-2α on TRAIL sensitivity in other cancer cell lines
(a) Three pancreatic cancer cell lines (MiaPaca-2, SW1990, and Panc10.05), a prostate cancer cell line (DU145), and a cervical cancer cell line (HeLa) were transfected with the indicated siRNAs and cultured under hypoxic conditions (1% O2) for 24 h. Then, nuclear extracts of the cells were subjected to immunoblotting. E2F-1 was used as a loading control. (b) siRNA-transfected cancer cells were cultured with TRAIL under hypoxic conditions for 48 h. Cell viability was analyzed using the WST-8 assay. The results are presented as means ± SD from triplicate experiments. *p < 0.05, **p < 0.01 compared to negative control siRNA.
Figure 4
Figure 4. TRAIL sensitivity of HIF-2α shRNA-expressing Panc-1cells both in vitro and in vivo
(a) The nuclear extracts of Panc-1 cells were used for immunoblotting. E2F-1 was used as a loading control. N: normoxic, H: hypoxic (1% O2 for 24 h), D: deferoxamine mesylate (100μM, normoxic for 24 h). (b) shRNA-expressing Panc-1 cells were treated with TRAIL at the indicated concentrations under hypoxia or normoxia for 48 h. Cell viability was assessed using the WST-8 assay. **p < 0.01 was compared to control shRNA-transduced cells. Similar results were obtained from three independent experiments. (c) shRNA-expressing Panc-1 cells were incubated with 100 ng/mL TRAIL for 24 h and analyzed using flow cytometry. The results of Annexin V+ cells (%) are presented as means ± SD from triplicate experiments. **p < 0.01 (d) BALB/c nu/nu mice were inoculated with the indicated Panc-1 cells (5 × 106) with Matrigel into the right upper and lower two sites. On days 14, 15, and 16, vehicle (upper) control and TRAIL (1 μg; lower) at a volume of 50 μL were injected into the tumor sites, respectively. Each group consisted of six mice. Similar results were obtained from two independent experiments. **p < 0.01.
Figure 5
Figure 5. HIF-2α dictates TRAIL sensitivity of Panc-1 cells by regulating survivin expression
(a) Panc-1 cells were transfected with control, HIF-1α, or HIF-2α siRNA and the whole cell protein was extracted. Equal amounts of protein (500 μg) were used for the Proteome Profiler Apoptosis Array. (b) Panc-1 and AsPC-1 cells were transfected with the indicated siRNA. After 72 h, the cell lysates were used for immunoblotting. The band density of survivin was normalized to β-actin. (c) Two HREs in the human survivin promoter are shown. (d) Panc-1 cells were pre-transfected with either control HIF-1α or HIF-2α siRNA. On the next day, cells were co-transfected with TransLucent Survivin Gene Promoter Reporter Vector and pGL4.74 (hRluc/TK) vector. After 24 h incubation, the ratio of firefly/renilla luciferase was determined. The results are presented as means ± SD from triplicate experiments. Similar results were obtained from three independent experiments. *p < 0.05, **p < 0.01. (e) ChIP assay was performed on Panc-1 cells transfected with siRNA against HIF-2α or control. Data represents means ± SD from triplicate results which were normalized to input DNA. *, p < 0.05. (f) Immunoblotting was performed using the protein lysates from HIF-2α shRNA-expressing Panc-1 cells that were pre-transfected with mock control or survivin plasmid. (g) HIF-2α shRNA-expressing Panc-1 cells transfected with GFP-encoding mock or survivin plasmid were treated with TRAIL (100 ng/mL) for 24 h. Flow cytometric analysis was performed. After gating on survivin-GFP+ cells, the percentages of Annexin V+ apoptotic cells were determined. The numbers represent the percentages of each susbset. Similar results were obtained from two independent experiments. (h) The results of Annexin V+ cells (%) are presented as means ± SD from triplicate experiments. **p < 0.01.
Figure 6
Figure 6. Survivin suppressant YM155 sensitizes Panc-1 cells to TRAIL both in vitro and in vivo
(a) Two cancer cell lines were treated with YM155 for 48 h. Cell viability was assessed using the WST-8 assay. Similar results were obtained from two independent experiments. (b) Whole cell lysates from Panc-1 cells treated with YM155 for 24 h were used for immunoblotting. β-actin was used as a control. (c) Panc-1 cells treated with YM155 (5 nM) and/or TRAIL (25 ng/mL) were examined using flow cytometric analysis. Results of Annexin V+ cells (%) are presented as means ± SD from triplicate experiments. **p < 0.01 was compared to untreated control. Similar results were obtained from two independent experiments. (d) BALB/c nu/nu mice were inoculated s.c. with Panc-1 cells (5 × 106) with Matrigel into the right flank; the mice were pooled and divided into 2 groups. The mice were injected intraperitoneally (i.p.) with YM155 (5 mg/kg) or DMSO as a vehicle control (50 μL) for consecutive 3 days. Each group contained three mice. Protein lysates from tumor tissues were used for immunoblotting. The band density of survivin was normalized to β-actin. *p < 0.05. (e) Similarly, BALB/c nu/nu mice were inoculated subcutaneously with Panc-1 cells (5 × 106). On day 14, the mice were pooled and divided into four groups. The mice were treated with i.p. injection of YM155 (5 mg/kg) or DMSO as a vehicle control (50 μL) on days 14, 15, and 16. Each group contained six mice. After grouping, mice were treated with i.p. injection of YM155 (5 mg/kg) for 5 consecutive days (on days 14, 15, 16, 17, and 18) and with or without i.t. injection of TRAIL (1 μg) on days 16 and 18. Each group consisted of six mice. **p < 0.01 compared to vehicle control group. Similar results were obtained from two independent experiments.
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