Ougolkov, Andrei V.

Abstract Chronic lymphocytic leukemia (CLL) represents one of the most common hematological malignancies and is characterized by the expansion of a clonal B cell population. Commonly, CLL is defined as a disease of failed apoptosis of the leukemic B cells, and it is this feature that makes them refractory to common chemotherapeutic agents designed to kill CLL cells. The mechanism controlling apoptosis in CLL is complex and influenced by numerous factors, including NFkB-mediated expression of antiapoptotic proteins, such as Bcl-2 and XIAP. We recently demonstrated that glycogen synthase kinase-3 (GSK-3) b positively regulates NFkB-mediated gene transcription and cell survival in pancreatic cancer cells (Cancer Research 2005. 65:2076). Similar to pancreatic cancer, we have found that pharmacological inhibition of GSK-3 results in decreased expression of NFkB target genes Bcl-2, Mcl-1 and XIAP in CLL B cells. In addition we noted a dose dependent increase in apoptosis levels for both the MEC1 cell line and primary CLL B cell patient samples. Using CLL blood B cells, we have found that both GSK-3b and NFkB accumulate in the nucleus of CLL cells, but not normal blood B cells. In addition, we observed that inhibition of GSK-3 leads to a decrease in NFkB-mediated gene transcription, without affecting the nuclear accumulation of NFkB. However, using chromatin immunoprecipitation, we found that NFkB binding to its target gene promoters (XIAP, Bcl-2) is significantly affected in CLL B cells treated with a GSK-3 inhibitor. These results suggest that GSK-3 regulates the nuclear activity of NFkB in CLL cells by affecting the binding of p65/p50 to the promoters of a subset of its target genes. Our results establish that inhibition of GSK-3 induces apoptosis of CLL cells and suggest GSK-3b as a potential therapeutic target in the treatment of CLL.

Abstract Purpose: Enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in transcriptional repression through chromatin remodeling. Our objectives were to determine the expression pattern of EZH2 and to assess the anticancer effect of EZH2 depletion in pancreatic cancer cells. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the expression pattern of EZH2 in normal pancreas and human pancreatic tumors. We used RNA interference, Western blotting, reverse transcription-PCR, and chromatin immunoprecipitation to study the effect of EZH2 depletion on pancreatic cancer cell proliferation and survival. Results: We detected nuclear overexpression of EZH2 in pancreatic cancer cell lines and in 71 of 104 (68%) cases of human pancreatic adenocarcinomas. EZH2 nuclear accumulation was more frequent in poorly differentiated pancreatic adenocarcinomas (31 of 34 cases; P < 0.001). We found that genetic depletion of EZH2 results in reexpression of p27Kip1 and decreased pancreatic cancer cell proliferation. Moreover, we showed that EZH2 depletion sensitized pancreatic cancer cells to doxorubicin and gemcitabine, which leads to a significant induction of apoptosis, suggesting that the combination of EZH2 inhibitors and standard chemotherapy could be a superior potential treatment for pancreatic cancer. Conclusions: Our results show nuclear accumulation of EZH2 as a hallmark of poorly differentiated pancreatic adenocarcinoma; identify the tumor suppressor p27Kip1 as a new target gene of EZH2; show that EZH2 nuclear overexpression contributes to pancreatic cancer cell proliferation; and suggest EZH2 as a potential therapeutic target for the treatment of pancreatic cancer.

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.