Billadeau, Daniel D.

Abstract Chronic lymphocytic leukemia (CLL) is commonly defined as a disease of failed apoptosis of B cells and remains an incurable disease. The mechanism of resistance to apoptosis in CLL is complex and influenced by numerous factors, including nuclear factor κB (NFκB)-mediated expression of antiapoptotic molecules. Recent evidence indicates that glycogen synthase kinase-3β (GSK-3β) positively regulates NFκB-mediated gene transcription and cell survival. Using malignant B cells collected from patients with CLL, we find that both GSK-3β and NFκB accumulate in the nucleus of CLL B cells, and pharmacologic inhibition of GSK-3 results in decreased expression of two NFκB target genes Bcl-2 and XIAP and a subsequent increase in CLL B-cell apoptosis ex vivo. Furthermore, we observed that inhibition of GSK-3 leads to a decrease in NFκB-mediated gene transcription but does not affect the nuclear accumulation of NFκB in CLL B cells. Last, using chromatin immunoprecipitation, we show that GSK-3 inhibition abrogates NFκB binding to its target gene promoters (XIAP, Bcl-2), in part through epigenetic modification of histones. Our results establish that inhibition of GSK-3 abrogates NFκB binding to its target gene promoters through an epigenetic mechanism, enhances apoptosis in CLL B cells ex vivo and identifies GSK-3 as a potential therapeutic target in the treatment of CLL.

Abstract Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) β regulates nuclear factor-κB (NF-κB)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3β in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3β affects NF-κB activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3β in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Results: We found nuclear accumulation of GSK-3β in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3β nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3β can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-κB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts. Conclusions: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3β nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3β regulates NF-κB activity in pancreatic cancer.

Abstract Recent studies using glycogen synthase kinase-3β (GSK-3β)–deficient mouse embryonic fibroblasts suggest that GSK-3β positively regulates nuclear factor κB (NFκB)–mediated gene transcription. Because NFκB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3β in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3β and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3β by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3β influences NFκB-mediated gene transcription at a point distal to the Iκ kinase complex, as only ectopic expression of the NFκB subunits p65/p50, but not an Iκ kinase β constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3β inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3β in cancer cell survival and proliferation and suggest GSK-3β as a potential therapeutic target in the treatment of pancreatic cancer.