Differential activity of GSK-3 isoforms regulates NF-κB and TRAIL- or TNFα induced apoptosis in pancreatic cancer cells

Abstract

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3α or GSK-3β. In contrast, depletion of GSK-3β, but not GSK-3α, sensitized PDA cell lines to TNFα-induced cell death. Further experiments demonstrated that TNFα-stimulated IκBα phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3β-deficient MEFs. Nonetheless, inhibition of GSK-3β function in MEFs or PDA cell lines impaired the expression of the NF-κB target genes Bcl-xL and cIAP2, but not IκBα. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3β targeted to the nucleus but not GSK-3β targeted to the cytoplasm, suggesting that GSK-3β regulates NF-κB function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3β overexpression and nuclear localization contribute to TNFα and TRAIL resistance via anti-apoptotic NF-κB genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

Figure 1

GSK-3 inhibition enhances both TNFα- and TRAIL-induced apoptosis in pancreatic cancer cells. (a, b) Cell proliferation determined by MTS assay. Pancreatic cancer cells were pretreated for 30 min with 0.2 μM of GSK-3i (LY2064827) or diluent (0.1% DMSO) followed by co-treatment with the indicated concentrations of TNFα or TRAIL for 24 h. (c–f) Cells were treated with diluent, GSK-3i and TNFα or TRAIL for 24 h as in (a) and dually stained with annexin V-FITC and PI for flow cytometry. (e, f) Results from three independent experiments were quantified and shown as mean±S.D. with indicated P-values (Student's t-test). (g) HupT3 cells were treated with GSK-3i and TNFα alone or in combination for 24 and 48 h, respectively. Whole-cell extracts were prepared for immunoblot analysis for PARP1. (h, i) Indicated pancreatic cancer cells were treated with GSK-3i (0.2 μM) and TRAIL (2 ng/ml) alone or in combination for 24 h. Whole-cell extracts were immunoblotted with antibodies specific for PARP1 and cleaved CASP3. Arrows indicate cleaved PARP1 (g–i)

Figure 2

Isoform-specific role of GSK-3 in TRAIL- and TNFα-induced apoptosis. (a) Whole-cell extracts prepared from cells stably transduced with isoform-specific lentiviral shRNAs or scrambled control shRNA were subjected to immunoblotting to demonstrate specific and efficient knockdown of endogenous GSK-3 proteins (upper panel). Lentiviral shRNA transduced cells were treated with TRAIL or TNFα for 24 h. Whole-cell extracts were subjected to immunoblot against PARP1 and cleaved CASP3 (lower panel). Arrow indicates cleaved PARP1. (b, c, f) Panc04.03 stably transduced with lentiviral shRNA (b) or MEFs (c) were treated with TNFα(20 and 2 ng/ml, respectively) for 18 h and stained with annexin V-FITC and PI for flow cytometry. (d, e, g) Cells stably transduced with the indicated shRNA were treated with TRAIL (5 ng/ml) or diluent for 18 h, stained with annexin V-FITC/PI and examined by flow cytometry. (f, g) Results from three independent experiments were quantified and shown as mean±S.D. with indicated P-values (Student's t-test). Note that only GSK-3β knockdown or deletion enhanced TNFα-induced apoptosis in Panc04.03 or MEF cells (f), whereas knockdown of either GSK-3α or GSK-3β sensitized the cells equally to TRAIL (g)

Figure 3

GSK-3β is not essential for TNFα-induced IKBα degradation or p65 nuclear translocation. (a, b) Western blot analysis of whole-cell extracts (a) or cytosolic and nuclear extracts (b) from MEFs treated with 10 ng/ml TNFα for the indicated periods of time. Note that there is no discernible difference in TNFα- induced IκBα phosphorylation/degradation (a) or p65 nuclear translocation (b) between WT and GSK-3β-null MEF cells. (c) Immunofluorescence staining and confocal microscopic imaging showing p65 expression/localization in MEF cells following TNFα stimulation (10 ng/ml) for the indicated periods of time. (d) Quantification of images (c) for percentage of p65 with predominant cytoplasmic or nuclear localization in wild-type (WT) and Gsk-3β^−/− (KO) MEF cells at the indicated time points. Arrows point to the cells shown in inset with p65 predominantly localized to the cytoplasm (0 min) or nucleus (20 and 60 min). (e) Confocal microscopic imaging showing in situ p50 and p65 interaction detected by PLA following TNFα stimulation. (f) Quantification of dots corresponding to sites of interaction as shown in (e) as mean±S.D. as described in Materials and Methods

Figure 4

GSK-3 inhibition and GSK-3β suppression regulate a subset of anti-apoptotic NF-κB target genes. (a, b) Western blot analysis of whole-cell extracts (a) or cytosolic and nuclear fractions (b) with indicated antibodies. BxPC-3 cells were treated for 24 h with the indicated concentrations of GSK-3i before preparation of protein extracts. (c) Luciferase reporter assay in BxPC-3 and HEK293T cells stably transduced with TopFlash and treated with the indicated concentration of GSK-3i for 6 h. The relative firefly luciferase activities were normalized to renilla luciferase activity and plotted as a histogram. (d) After Panc04.03 cells were treated with 0.5 μM of GSK-3i for 24 and 48 h, respectively, whole-cell extracts were prepared and immunoblotted with the indicated antibodies. (e) Whole-cell extracts from Panc04.03 cells stably transduced with GSK-3α or GSK-3β lentiviral shRNA were probed with the indicated antibodies

Figure 5

GSK-3β differentially affects the binding of NF-κB p65 and p50 to the BCL2L1 and BIRC3 promoters. (a, b) qRT-PCR analysis of MEF and Panc04.03 cells following TNFα-stimulation. Note that Bcl-xL and cIAP2 transcripts, but not IKBα, were reduced in Gsk-3β-null MEFs compared with WT control (a). Similarly, Bcl-xL and cIAP2, but not IKBα mRNA expression was reduced in TNFα-treated Panc04.03 cells transduced with lentiviral GSK-3β shRNA (b). (c–e). ChIP analysis of Panc04.03 cells treated with GSK-3i (0.5 μM LY2064827) or diluent (0.1% DMSO) for 16 h. The relative amount of DNA precipitated with the specified antibodies was accessed by qPCR using three sets of primers targeting promoter regions containing the depicted NF-κB sites. The qPCR was performed in triplicate; and the result is normalized to diluent control

Figure 6

Nuclear GSK-3β is active and contributes to regulation of Bcl-xL and cIAP2. (a) Western blot analysis of cytosolic and nuclear fractions from the indicated cell lines. Note the abundant presence of nuclear GSK-3β and minimal S9 phosphorylation in all pancreatic cancer cells. (b) Immunofluorescence staining and confocal microscopic imaging showing the expression/localization of GSK-3β in Panc04.03. Cells were transfected for 48 h with pCMS4-eGFP-H1P suppression/re-expression vectors for the expression of C-terminal Flag-tagged GSK-3β including WT, S9A mutant, and S9A with NLS and NES signals. The plasmid also expresses EGFP driven by an independent promoter, making it feasible to identify individual transfected cells. (c) Cytosolic and nuclear protein samples from above transfected cells were subjected to immunoblotting with indicated antibodies. ORC2 is used as a marker for nuclear protein. (d) qRT-PCR showing effect of reconstituted GSK-3β expression on Bcl-xL, cIAP2 and IκBα expression. Results of one representative experiment are shown as mean of triplicates ±S.D. and normalized to RPLP0 expression

Figure 7

Bcl-xL and XIAP participate in GSK-3i-induced sensitization to TRAIL/ and TNFα. (a) Representative flow cytometric profile of annexin V-FITC and PI stained HupT3 cells to evaluate apoptosis induction. HupT3 cells with stable expression of lentiviral shRNA for Bcl-xL, cIAP2 or XIAP (or scrambled control) were treated with diluent, TNFα or TRAIL as indicated for 18 h before harvesting and staining. (b) Summarized results from three independent analyses as percentage of annexin V-FITC and/or PI cells to total gated cells were shown as average±S.D. (c, d) Whole-cell extracts from above HupT3 cells (c) and BxPC-3 cells stably re-expressing Flag.Bcl-xL (d) were subjected to immunoblotting to confirm efficient suppression of endogenous gene expression and rescued Bcl-xL expression. (e) BxPC-3 cells with stable lentiviral-mediated expression of Flag.Bcl-xL were treated and stained as in (a) for apoptosis analysis