Recent studies using glycogen synthase kinase-3B (GSK-3B)-deficient mouse embryonic fibroblasts suggest that GSK-3B positively regulates nuclear factor KB (NFKB)-mediated gene transcription. Because NFKB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3B in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3B and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3B by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3B influences NFKBmediated gene transcription at a point distal to the IK kinase complex, as only ectopic expression of the NFKB subunits p65/p50, but not an IK kinase B constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3B inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3B in cancer cell survival and proliferation and suggest GSK-3B as a potential therapeutic target in the treatment of pancreatic cancer. (Cancer Res 2005; 65(6): 2076-81)

GSK3β is a multifunctional serine/threonine kinase that regulates various cellular pathways, depending on its substrates for phosphorylation. It is evident that regulation of Wnt/β-catenin signaling is only one of its diverse functions. Since oncogenic transcription factors (e.g., c-Jun, c-Myc) and proto-oncoproteins (i.e., β-catenin, Gli proteins) are putative GSK3β substrates for phosphorylation-dependent inactivation, it is hypothesized that GSK3β interferes with cellular neoplastic transformation and tumor development, as exemplified by its activity in Wnt/β-catenin signaling. However, only a few studies have addressed its role(s) in human cancer, and these studies have reported differing effects of GSK3β on cancer cells. Using GSK3β deficient mouse embryonic fibroblasts, it was shown that GSK3β plays a crucial role in cell survival mediated by the nuclear factor-kappaB (NF-κB) pathway (Nature 2000; 406:86-90). Interestingly, we have recently shown that the Wnt/β-catenin and NF-κB pathways were co-activated in colorectal cancer by dysregulation in the ubiquitin system (J Natl Cancer Inst 2004; 96:1161-70). Thus, these observations bring forward apparently opposing notions regarding the functions of GSK3β in neoplastic cells on the one hand, removing a neoplastic trigger by phosphorylation-dependent degradation of β-catenin oncoprotein in the ubiquitin system, and on the other, contributing to a cell proliferation and survival pathways by regulating NF-κB. The present study was therefore undertaken to clarify the role of GSK3β in cancer by analyzing expression and activity of this kinase in colon cancer cells and clinical colorectal cancers and by investigating its effects on cancer cells. In colon cancer cell lines and colorectal cancer patients, levels of GSK3β expression and its active form were higher in tumor cells than in their normal counterparts; these findings were independent of nuclear accumulation of β-catenin oncoprotein in the tumor cells. Inhibition of GSK3β activity by its Ser9 phosphorylation was defective in colorectal cancers but preserved in non-neoplastic cells and tissues. Strikingly, inhibition of GSK3β activity by chemical inhibitors and its expression by RNA interference targeting GSK3β induced apoptosis and attenuated proliferation of colon cancer cells in vitro. Our findings demonstrate an unrecognized role of GSK3β in tumor cell survival and proliferation and warrant proposing this kinase as a novel and potential therapeutic target in colorectal cancer.

Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that beta-catenin stabilizes the mRNA encoding the F-box protein betaTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of beta-catenin/Tcf transcription factor. CRD-BP binds to the coding region of betaTrCP1 mRNA. Overexpression of CRD-BP stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)(betaTrCP) E3 ubiquitin ligase and in accelerated turnover of its substrates including IkappaB and beta-catenin. CRD-BP is essential for the induction of both betaTrCP1 and c-Myc by beta-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active beta-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of betaTrCP1, in the activation of dimeric transcription factor NF-kappaB and in the suppression of apoptosis in these cancers.

Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) h regulates nuclear factor-nB (NF-nB)^mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3h 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-3h affects NF-nB activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3h 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-3h in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3h nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3h 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-nB-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-3h nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3h regulates NF-nB activity in pancreatic cancer.Despite tremendous scientific efforts, conventional treatment approaches have had little effect on the course of pancreatic cancer. There are numerous factors that contribute to progression of this disease, including constitutively active nuclear factor-nB ref. 1). Activation of NF-nB in human cancer has been shown to positively influence cancer cell survival, proliferation, invasion, metastasis, and chemoresistance (2, 3). Thus, the identification of the altered molecular pathways regulating NF-nB activity is a major focus of cancer researchers, as these studies will provide valuable knowledge and identify novel targets to antagonize NF-nB activation in human cancer.The cytoplasmic serine/threonine protein kinase glycogen synthase kinase-3 (GSK-3) was first described as a component of the metabolic pathway for glycogen synthase regulation (4). There are two homologous mammalian isoforms encoded by different genes, . Surprisingly, similar to the disruption of the NF-nB p65 or InB kinase h genes, ablation of the murine GSK-3h gene resulted in embryonic lethality due to hepatocyte apoptosis and massive liver degeneration (6 -8). These findings suggest a role for GSK-3h (but not GSK-3a) in the mechanism of ...

IntroductionChronic lymphocytic leukemia (CLL) is the most common human hematologic malignancy and, despite substantial scientific efforts, remains an incurable disease. Whereas some patients with CLL have a slow course of disease, most face inevitable progression and have fatal outcomes. 1,2 CLL is characterized by the accumulation of largely nonproliferating leukemic B cells that are resistant to apoptosis. 3 An increasing body of evidence suggests that the apoptotic block of CLL B cells is linked to constitutively activated signaling pathways, including an active NFB pathway. 4,5 In fact, CLL B cells exhibit high constitutive levels of NFB activity compared with nonmalignant human B cells. 5 Because NFB regulates the expression of antiapoptotic molecules including Bcl-2 and XIAP, a sustained activation of NFB pathway is critical for the survival of CLL B cells. 6 Thus, identification of the altered pathways regulating NFB activity in CLL B cells may lead to the discovery of novel therapeutic targets to antagonize NFB activation and induce apoptosis in these leukemic B cells.Glycogen synthase kinase (GSK)-3, a serine/threonine protein kinase, was first described as a component of the metabolic pathway for glycogen synthase regulation. 7 Two homologous mammalian GSK-3 isoforms are encoded by different genes, GSK-3␣ and GSK-3␤. 8 It has been shown that, similar to the disruption of the NFB p65 or IB kinase ␤ (IKK␤) genes, ablation of the murine GSK-3␤ gene is lethal to embryos as a result of TNF␣-induced hepatocyte apoptosis and massive liver degeneration. 9-11 These findings suggest a role for GSK-3␤ (but not GSK-3␣) in the regulation of NFB activation. The early steps leading to NFB activation after tumor necrosis factor ␣ (TNF␣) treatment (degradation of IB␣ and translocation of NFB to the nucleus) were unaffected in GSK-3␤-deficient mouse embryonic fibroblasts (MEFs), indicating that NFB is regulated by GSK-3␤ at the level of the transcriptional complex. 11 Consistent with this idea, we have recently shown that GSK-3␤ participates in NFB-mediated pancreatic cancer cell survival and proliferation by regulating NFB activity at a point downstream of the activation of the IKK complex. 12 Taken together, these data rule out an effect of GSK-3␤ on the cascade of proteins that culminates in phosphorylation of IB␣ and its degradation and suggest that GSK-3␤ may regulate the nuclear activity of NFB p65/p50.Although CLL B cells exhibit high constitutive levels of NFB activity, 5 the localization of GSK-3␤ in human CLL B cells and whether GSK-3␤ affects NFB activity are unknown. In the present study, we find that GSK-3␤ accumulates in the nuclei of human CLL B cells. We demonstrate that pharmacologic inhibition of GSK-3 leads to depletion of its nuclear pool, suppression of NFB transcriptional activity, decreased expression of antiapoptotic proteins (XIAP, Bcl-2), and enhanced apoptosis in CLL B cells. From a mechanistic perspective, we provide evidence that inhibition of GSK-3␤ affects histone modification at two NFB ...

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 71of 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 p27 Kip1 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.

In colorectal cancer, increased expression of beta-TrCP1 is associated with activation of both beta-catenin and NF-kappaB, suggesting that the integration of these signaling pathways by increased beta-TrCP expression may contribute to an inhibition of apoptosis and tumor metastasis.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3b positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3b and to assess the anti-cancer effect of GSK-3b inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3b in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT -PCR, BrDU incorporation and MTS assays to study the effect of GSK-3b inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3b in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3b as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.