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.

Glycogen synthase kinase-3β regulates tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis via the NF-κB pathway in hepatocellular carcinoma

“…TRAIL has been reported to activate the transcription factor NF-κB, which transactivates genes encoding the key anti-apoptotic proteins Bcl-xL and IAPs, both of which have been implicated in TRAIL resistance (10,11). The current findings demonstrated that GSK-3β inhibitor sensitizes HCC cells to TRAIL-induced apoptosis, suggesting that GSK-3β may be involved in the regulation of NF-κB activation.…”

“…In addition, Bcl-2 was reported to inhibit TRAIL-mediated cell death (27). IAPs constitute a protein family that regulates programmed cell death (37), and a recent study demonstrated that GSK-3β induces Bcl-xL and cIAP2 expression in pancreatic cancer cells (11). Consistent with this, the results of the present study indicate that GSK-3β may also regulate the expression of the BCL2L1 and BIRC3 genes, which encode anti-apoptosis proteins Bcl-xL and cIAP2 in HCC cells, respectively, and are known downstream targets of NF-κB.…”

“…A recent study revealed that Bcl-xL downregulation contributes to GSK-3β inhibitor-induced sensitization to TRAIL in pancreatic ductal adenocarcinoma cells (11). To evaluate the role of Bcl-xL and cIAP2 on TRAIL-induced apoptosis in HCC cells, shRNA was used to generate HuH-7 cells with stable knockdown of each gene.…”

“…Previous studies have demonstrated that TRAIL can activate the NF-κB signaling pathway, which activates genes that encode various key anti-apoptotic proteins, such as B-cell lymphoma-extra large (Bcl-xL) and inhibitor of apoptosis proteins (IAPs). These proteins contribute to TRAIL resistance (10,11). Thus, overcoming NF-κB-associated survival signals may enhance the antitumor effect of TRAIL in HCC cells.…”

TRAIL Mediated Signaling in Pancreatic Cancer

“…However, Bclxl knockdown cells reconstituted with Bcl-xl, indicated partial reversal of GSK-3 inhibitor and TRAIL-induced apoptosis. Mechanistically it was reported that GSK-3i treatment resulted in an increased SIRT1 and HDAC3 loading at BCL2L1 promoter that consequently resulted in formation of repressive chromatin (Zhang et al, 2014). Embelin, isolated from the fruit of Embelia ribes has been shown to inhibit growth of AsPC-1 xenografts in Balb C nude mice.…”