The long non-coding RNA HOTAIR enhances pancreatic cancer resistance to TNF-related apoptosis-inducing ligand

Abstract

Pancreatic cancer is a malignant neoplasm with a high mortality rate. Therapeutic agents that activate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis have shown promising efficacy, but many pancreatic cancers are resistant to TRAIL therapy. Epigenetic regulation plays important roles in tumor pathogenesis and resistance, and a recent study indicated that the long non-coding RNA HOX transcript antisense RNA (HOTAIR) is overexpressed in pancreatic cancer. However, the role of HOTAIR in pancreatic cancer resistance to anticancer agents is unknown. The present study determined the role of HOTAIR in pancreatic cancer TRAIL resistance and investigated the underlying molecular mechanisms. We observed that TRAIL-resistant pancreatic cancer cells had higher levels of HOTAIR expression, whereas TRAIL-sensitive pancreatic cancer cells had lower HOTAIR levels. Overexpressing HOTAIR in TRAIL-sensitive cells attenuated TRAIL-induced apoptosis, and shRNA-mediated HOTAIR knockdown in TRAIL-resistant PANC-1 cells sensitized them to TRAIL-induced apoptosis. These results support a causative effect of HOTAIR on TRAIL sensitivity. Mechanistically, we found that increased HOTAIR expression inhibited the expression of the TRAIL receptor death receptor 5 (DR5), whereas HOTAIR knockdown increased DR5 expression. We further demonstrated that HOTAIR regulates DR5 expression via the epigenetic regulator enhancer of zeste homolog 2 (EZH2) and that EZH2 controls histone H3 lysine 27 trimethylation on the DR5 gene. Taken together, these results demonstrate that high HOTAIR levels increase the resistance of pancreatic cancer cells to TRAIL-induced apoptosis via epigenetic regulation of DR5 expression. Our study therefore supports the notion that targeting HOTAIR function may represent a strategy to overcome TRAIL resistance in pancreatic cancer.

Keywords: DR5; TRAIL; apoptosis; death receptor 5; histone methylation; long non-coding RNA (long ncRNA, lncRNA); pancreatic cancer.

Figure 1.

HOTAIR expression in pancreatic cancer cells with different sensitivity to TRAIL-induced apoptosis. A, HOTAIR expression in pancreatic cell lines, BxPC3, MiaPaCa-2, Suit2, and PANC-1, as determined by qRT-PCR and normalized by the expression of β-actin. Results shown are means ± S.D. of three independent experiments performed in duplicate. B, TRA-8-induced apoptosis. Pancreatic cancer cells were exposed to TRA-8 (1 μg/ml) for 24 h; and apoptotic cells were detected by flow cytometry (n = 3, *, p < 0.05, ***, p < 0.001).

Figure 2.

Overexpression of HOTAIR attenuates TRA-8-induced apoptosis in sensitive pancreatic cancer cells. A and B, BxPC3 (A) and MiaPaCa-2 (B) cells were infected with lentiviruses carrying control vector or HOTAIR cDNA (HOTAIR), and stable clones were selected by puromycin. Panels Aa and Ba, HOTAIR expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, ***, p < 0.001). Panels Ab and Bb, TRA-8-induced apoptosis. BxPC3 and MiaPaCa-2 cells with HOTAIR overexpression and their control vector cells were seeded into 6-well plates at 2 × 10⁵ per well. After culturing for 24 h, cells were exposed to TRA-8 (1 μg/ml) for 24 h, and apoptosis was determined by flow cytometry using Annexin PE and 7 AAD staining kit. TRA-8-induced apoptosis is shown in the hatched bars (n = 3, ***, p < 0.001). Panels Ac and Bc, Western blot analysis of the expression of caspase-8 (Casp8). The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown.

Figure 3.

Down-regulation of HOTAIR promotes TRA-8-induced apoptosis in resistant pancreatic cells. A and B, PANC-1 (A) and Suit2 (B) cells were infected with lentiviruses carrying scrambled shRNA (shScr) or shRNA for HOTAIR (shHOTAIR) and selected by puromycin. Panels Aa and Ba, HOTAIR expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, **, p < 0.01, ***, p < 0.001). Panels Ab and Bb, TRA-8-induced apoptosis. Cells were exposed to TRA-8 (1 μg/ml) for 24 h, and apoptosis was determined by flow cytometry. TRA-8-induced apoptosis is shown in the hatched bars (n = 3, **, p < 0.01, ***, p < 0.001).

Figure 4.

HOTAIR regulates DR5 expression in pancreatic cancer cells. A and B, overexpression of HOTAIR inhibited DR5 expression BxPC3 and MiaPaCa-2 cells stably expressing HOTAIR cDNA (HOTAIR) or control vector were selected by puromycin. Panels Aa and Ba, DR5 mRNA expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, **, p < 0.01). Panels Ab and Bb, DR5 protein expression, as determined by Western blot analysis. The expression of β-actin expression was used as a loading control. Representative blots from three independent experiments are shown. C and D, HOTAIR knockdown increased DR5 expression. PANC-1 and Suit2 cells stably expressing scrambled shRNA (shScr) or shRNA for HOTAIR (shHOTAIR) were selected by puromycin. Panels Ca and Da, DR5 mRNA expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, *, p < 0.05, **, p < 0.01). Panels Cb and Db, DR5 protein expression, as determined by Western blotting. The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown.

Figure 5.

Inhibition of EZH2 increases TRA-8-induced apoptosis. A, DZNeP increased TRA-8-induced apoptosis in resistant cells. PANC-1 (panel a) and Suit2 (panel b) cells were exposed to DZNeP (5 μm), an EZH2 inhibitor, and subsequently treated with TRA-8 (1 μg/ml) for 24 h. Apoptosis was determined by flow cytometry (n = 3, ***, p < 0.001). B, DZNeP attenuated HOTAIR-overexpression-induced TRA-8 resistance. BxPC3 (panel a) and MiaPaCa-2 (panel b) cells with stably HOTAIR overexpression were exposed to DZNeP (5 μm) and subsequently treated with TRA-8 (1 μg/ml) for 24 h. Apoptosis was determined by flow cytometry (n = 3, *, p < 0.05, **, p < 0.01). C and D, EZH2 knockdown enhanced DR5 expression and TRA-8-induced apoptosis. PANC-1 and Suit2 cells stably expressing scrambled shRNA (shScr) or shRNA for EZH2 (shEZH2) were selected by puromycin. Panels Ca and Da, cells were exposed to TRA-8 (1 μg/ml) for 24 h; apoptosis was determined by flow cytometry (n = 3, *, p < 0.05, ***, p < 0.001). Panels Cb and Db, Western blot analyses of the expression of EZH2 and DR5. The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown.

Figure 6.

HOTAIR regulates DR5 expression via EZH2-mediated modulation of histone H3 lysine 27 trimethylation (H3K27me3) on DR5 gene. A, DZNeP inhibited H3K27me3 in pancreatic cancer cells. PANC-1 cells were exposed to DZNeP (5 μm) for 24 h. H3K27me3 and DR5 expression was determined by Western blot analysis. The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown. B, HOTAIR knockdown inhibited H3K27me3 on DR5 gene. PANC-1 cells stably expressing scrambled shRNA (shScr) or shRNA for HOTAIR (shHOTAIR) were selected by puromycin. Panel a, Western blot analyses of EZH, DR5, and H3K27me3. Panels b and c, ChIP was performed using mouse anti-trimethyl-histone 3 (lysine 27) antibody or control mouse IgG. H3K27me3-bound DR5 gene was determined by PCR (panel b) and quantitative PCR (panel c). Representative results of three independent experiments are shown. *, p < 0.05. C, HOTAIR overexpression increased H3K27me3 on DR5 gene. MiaPaCa-2 cells stably expressing HOTAIR cDNA or control vector were selected by puromycin. Panel a, Western blot analysis of the expression of EZH2, DR5, and H3K27me3. The expression of β-actin was used as a loading control. Panels b and c, ChIP analysis of H3K27me3 on DR5 gene, determined by PCR (panel b) and quantitative PCR (panel c). Representative results of three independent experiments are shown. *, p < 0.05. D, schematic model of HOTAIR/EZH2-mediated H3K27me3 and DR5 expression in pancreatic cancer cells.