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Human Cancer Biology |
Authors' Affiliations: 1 Division of Oncology Research, 2 Gastrointestinal Unit, 3 Department of Laboratory Medicine and Pathology, and 4 Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota and 5 Department of Urology, Yamagata University School of Medicine, Yamagata, Japan
Requests for reprints: Daniel D. Billadeau, Department of Immunology and Division of Oncology Research, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-266-4334; Fax: 507-266-5146; E-mail: billadeau.daniel{at}mayo.edu.
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Abstract |
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B (NF-B)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3ß 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-3ß affects NF-B activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3ß 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-3ß in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3ß nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3ß 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-B-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-3ß nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3ß regulates NF-B activity in pancreatic cancer.
B (NF-B; ref. 1). Activation of NF-B 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-B activity is a major focus of cancer researchers, as these studies will provide valuable knowledge and identify novel targets to antagonize NF-B 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, GSK-3
and GSK-3ß (5). Surprisingly, similar to the disruption of the NF-B p65 or IB kinase ß genes, ablation of the murine GSK-3ß gene resulted in embryonic lethality due to hepatocyte apoptosis and massive liver degeneration (6–8). These findings suggest a role for GSK-3ß (but not GSK-3) in the mechanism of NF-B activation and suggest that GSK-3ß may be a potential therapeutic target in human cancer. Using GSK-3ß-deficient mouse embryonic fibroblasts, it was shown that the early steps leading to NF-B activation following TNF- treatment (degradation of IB and translocation of NF-B to the nucleus) were unaffected by the loss of GSK-3ß, indicating that NF-B is regulated by GSK-3ß at the level of the transcriptional complex (8). Consistent with this idea, we have shown recently that GSK-3ß participates in NF-B-mediated pancreatic cancer cell survival and proliferation in vitro by regulating NF-B activity at a point downstream of the activation of the IB kinase complex (9). Taken together, these data rule out an effect of GSK-3ß on the cascade of proteins that culminates in phosphorylation of IB and its degradation and suggest that GSK-3ß may regulate the nuclear activity of NF-B p65/p50. However, whether GSK-3ß can be accumulated in the nuclei of cancer cells where it can contribute to NF-B transcriptional activity is not known.
The localization of GSK-3ß in human cancer cells and the mechanism by which GSK-3ß affects NF-B activity has not yet been determined. Here, we find that GSK-3ß is overexpressed in human pancreatic tumors and accumulates in the nuclei of pancreatic cancer cell lines and most poorly differentiated pancreatic adenocarcinomas. Additionally, we show that nuclear accumulation of GSK-3ß is dependent on its kinase activity and pharmacologic inhibition of GSK-3 leads to a loss of GSK-3ß from the nucleus of pancreatic cancer cells. Furthermore, for the first time, we show that inhibition of GSK-3 affects NF-B-mediated survival and proliferation of cancer cells in established tumor xenografts and suppresses pancreatic tumor growth in vivo.
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Materials and Methods |
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Immunoblot analysis and antibodies. For immunoblots, cells were lysed as described previously (9). Nuclear/cytosolic fractionation was done by Dignam method (11). Nuclear cell lysate was treated with DNase I (1 unit/µL) to obtain total histone H3 (nuclear marker). Whole-protein extract from tumor tissue was prepared as described (12). Protein sample concentration was quantified and equal amount (50 µg whole, nuclear, or cytosolic protein extract) of protein was loaded in each well of SDS-polyacrylamide gel. Cell or tissue extracts were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed as indicated. The following antibodies were used for immunoblot analysis: GSK-3ß, ß-catenin, cyclin D1, Bcl-2, X-linked inhibitor of apoptosis protein (XIAP), and ORC2 (BD PharMingen, San Diego, CA); NF-B p65 (Santa Cruz Biotechnology, Santa Cruz, CA); histone H3 (Abcam, Cambridge, MA); and Cu/Zn superoxide dismutase (Stressgen, Victoria, British Columbia, Canada). Bound antibodies were detected as described previously (9).
Analysis of apoptosis and bromodeoxyuridine staining. Carcinoma cells undergoing apoptosis were detected in xenograft tumor tissue sections by the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) method (13). The apoptotic index of each tumor was calculated as described previously (12). Bromodeoxyuridine incorporation assay was carried out as described (9, 14).
Immunohistochemistry. All studies carried out on human specimens were approved by the Institutional Review Board at the Mayo Clinic (Rochester, MN). The following antibodies were used for immunostaining: GSK-3ß and ß-catenin; cyclin D1 (Biocare Medical, Concord, CA); Bcl-2 (DakoCytomation, Glostrup, Denmark); XIAP (R&D Systems, Minneapolis, MN); phosphorylated Ser10 histone H3 (Upstate, Lake Placid, NY); and NF-B p65. GSK-3ß immunostaining was done on 122 resected primary pancreatic adenocarcinoma specimens (54 females and 68 males, mean age, 62.7 ± 1). Two pathologists (A.V.O. and T.C.S.) independently reviewed all cases and classified the tumors as well differentiated (n = 22), moderately differentiated (n = 59), or poorly differentiated (n = 41). For each case, the most representative section reflecting the major features of the primary pancreatic tumor (i.e., histologic type) was selected for immunohistochemical examination to determine the expression of GSK-3ß. Pancreatic intraepithelial neoplasia (PanIN) lesion specimens were obtained from 47 patients and stained to detect GSK-3ß expression. Metastatic lymph node specimens were obtained from 10 pancreatic cancer patients and analyzed by immunohistochemistry for GSK-3ß, cyclin D1, and ß-catenin expression and NF-B activation, represented by its nuclear accumulation. Immunohistochemical staining was done as described (12). GSK-3ß expression in tumor cells was classified into three patterns as follows: (a) weak cytoplasmic expression similar to normal pancreatic acinar and ductal cells, (b) strong cytoplasmic expression, signified by significant increase of staining intensity in cytoplasm, and (c) nuclear accumulation, defined as staining of >10% of tumor cell nuclei regardless of intensity of cytoplasmic expression. Cyclin D1 and NF-B nuclear accumulation was defined as positive staining of >10% of cancer cell nuclei throughout the tumor. ß-Catenin expression was defined as any of three patterns: (a) membranous expression, similar to that in normal pancreatic ductal cells, (b) loss of membranous staining, signified by a loss of membranous expression found in >50% of cancer cells, and (c) nuclear accumulation, defined as staining of >10% of cancer cell nuclei throughout the tumor.
Statistical analysis. Data were analyzed using the Prism software package (GraphPad, Inc., San Diego, CA). Associations between GSK-3ß staining pattern and degree of tumor differentiation were analyzed using Fisher's exact test for 2 x 2 contingency tables or 
2 test for larger tables. Two-sided tests were used where applicable. 2 critical value for 0.05 probability level is 3.841, all 2 values exceeding 3.841 and Ps < 0.05 were considered to indicate statistical significance.
Xenograft tumor model. Female athymic nude mice (8-10 weeks old) were inoculated s.c. with 3 x 106 CAPAN2 pancreatic cancer cells mixed with Matrigel (BD Biosciences). Two weeks after injection, tumors were size matched and mice were randomized into two treatment groups: (a) control DMSO and (b) AR-A014418. Diluent (50 µL DMSO) or AR-A014418 (dissolved in 50 µL DMSO) was injected i.p. in a total volume of 500 µL PBS. Tumors were measured with calipers in three dimensions two times weekly. Tumor volume was calculated using the formula for volume of an ellipsoid: 4/3
x (L / 2) x (W / 2) x (H / 2), where L is the length, W is the width, and H is the height.
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Results |
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B activation at a point downstream of the activation of the IB kinase complex (9), suggesting a role for GSK-3ß in the regulation of NF-B transcriptional activity in pancreatic cancer cells. In view of these results, we sought to determine whether GSK-3ß accumulates in nuclei of human pancreatic cancer cells where it might contribute to NF-B transcriptional activity.
Using immunohistochemical staining for GSK-3ß, we found weak cytoplasmic GSK-3ß expression in normal human pancreatic ductal and acinar cells (Fig. 1A
). Weak cytoplasmic GSK-3ß staining of normal pancreatic ductal or acinar cells adjacent to tumor cells was used as an internal staining control. Similar to normal pancreatic ductal cells, weak cytoplasmic expression of GSK-3ß was observed in 16 of 18 and 4 of 12 cases of PanIN-1 and PanIN-2 lesions, respectively. GSK-3ß weak cytoplasmic staining was significantly related to PanIN-1 and PanIN-2 lesions [relative risk, 13.36; 95% confidence interval (95% CI), 7.240-24.67; odds ratio, 137.0; 95% CI, 27.95-671.4; P < 0.0001]. On the other hand, PanIN-3 lesions, well-differentiated adenocarcinomas (Fig. 1B), and moderately differentiated adenocarcinomas showed strong cytoplasmic expression of GSK-3ß in 16 of 17 (94%), 19 of 22 (86%), and 36 of 59 (61%) cases, respectively (Fig. 2A
). Significant association was observed between increased malignant phenotype of tumors from PanIN-1 to well-differentiated/moderately differentiated adenocarcinoma and shift from a weak to strong GSK-3ß cytoplasmic staining (2, 65.28; P < 0.0001).
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2, 44.13; P < 0.0001), with the highest expression rate in poorly differentiated adenocarcinomas (90%; relative risk, 2.387; 95% CI, 1.759-3.239; odds ratio, 38.11; 95% CI, 12.43-116.9; P < 0.0001). Our results suggest that induction of GSK-3ß overexpression occurs in PanIN lesions and increases toward differentiated adenocarcinomas, whereas nuclear accumulation of GSK-3ß is associated with the loss of pancreatic cancer differentiation, providing evidence that GSK-3ß nuclear accumulation is a late event in pancreatic tumorigenesis.
To investigate GSK-3ß overexpression and nuclear accumulation in metastatic pancreatic cancer cells, we analyzed metastatic lymph nodes from 10 pancreatic cancer patients for the expression and localization of GSK-3ß. Similar to the primary tissues, we found localization of GSK-3ß in the nuclei of pancreatic cancer cells in metastatic lymph nodes in 8 of 10 cases (Fig. 2B). Using immunostaining of serial sections, we observed NF-B nuclear accumulation in the same 8 specimens (Fig. 2C) and nuclear accumulation of cyclin D1, a NF-B target gene, was found in 10 of 10 cases (Fig. 2D). Membranous staining of ß-catenin (Fig. 2E) was observed in 8 of 10 cases. Taken together, these data suggest that nuclear accumulation of GSK-3ß is a common feature to both the primary pancreatic cancer and the metastatic disease.
Kinase activity correlates with GSK-3ß nuclear accumulation in pancreatic cancer cells. To determine whether nuclear accumulation of GSK-3ß was only a feature of primary pancreatic cancers, we analyzed GSK-3ß localization in pancreatic cancer cell lines and normal human pancreas tissue. Using nuclear/cytosolic fractionation, we found that all of the pancreatic cancer cell lines show cytoplasmic as well as nuclear localization of both GSK-3ß and p65 (Fig. 3A ). In contrast to these findings, GSK-3ß and p65 were not detected in the nuclear extract of normal human pancreatic cells (Fig. 3A). Thus, nuclear accumulation of GSK-3ß seems to be a feature of pancreatic cancer cells.
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Pharmacologic inhibition of GSK-3 suppresses NF-B-mediated pancreatic cancer cell proliferation and survival in established tumor xenografts. We have shown previously that pancreatic cancer cell lines are sensitive to the GSK-3 inhibitor AR-A014418 in vitro and that inhibition of GSK-3 leads to a reduction in the expression of several NF-B target genes, including Bcl-2, cyclin D1, and XIAP (9). To determine whether GSK-3 inhibition also leads to decreased cancer cell survival and proliferation through down-regulation of NF-B target genes in vivo, we injected either DMSO or 120 mg/kg AR-A014418 i.p. every 12 hours for 2 days into mice with established flank CAPAN2 xenografts (tumor volume, 350-400 mm3). Immunoblot analysis revealed a substantial decrease in polypeptide levels of the NF-B targets XIAP, Bcl-2, and cyclin D1 in tumor tissues from AR-A014418-treated animals (Fig. 4A
). In addition, as measured by TUNEL staining, AR-A014418-treated animals showed a 4-fold increase in the number of apoptotic cells compared with DMSO-treated animals as well as a 2-fold decrease in cancer cell proliferation (Fig. 4B and C). Using immunohistochemical staining, we found a pronounced decrease of histone H3 phosphorylation at Ser10 (a proliferation marker) in tumor xenografts treated with AR-A014418 (Fig. 4D and E). In addition, and consistent with the immunoblot analysis, we found cyclin D1 nuclear expression in 84 ± 19 versus 23 ± 7 cancer cells (mean number of positively stained cells per 100 counted cancer cells) in control and AR-A014418-treated animals, respectively (Fig. 4F and G). Moreover, immunohistochemical staining revealed a significant decrease in XIAP (Fig. 4H and I) and Bcl-2 (Fig. 4J and K) protein expression in tumors from AR-A014418-treated animals. These results suggest that pharmacologic inhibition of GSK-3 leads to inhibition of NF-B transcriptional activity and decreased pancreatic cancer cell proliferation and survival in established tumor xenografts.
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Discussion |
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B transcriptional activity and leads to decreased pancreatic cancer cell proliferation and survival in vitro (9). A recent series of studies have shown that inhibition of GSK-3ß also decreases proliferation and survival of colon (18), prostate (19), and hepatocellular cancer cells (20) and acute myeloid leukemia cells (21), thus identifying GSK-3ß as a potential therapeutic target in multiple human malignancies. Here, we find that GSK-3ß is overexpressed in human pancreatic tumors and accumulates in the nuclei of pancreatic cancer cell lines and most poorly differentiated pancreatic adenocarcinomas. Additionally, we show that GSK-3ß kinase activity contributes to its nuclear accumulation in pancreatic cancer cells. Furthermore, for the first time, we show that inhibition of GSK-3 suppresses pancreatic tumor growth in vivo and affects NF-B-mediated survival and proliferation of cancer cells in established tumor xenografts. Taken together, our results suggest GSK-3ß as a potential therapeutic target in the treatment of human pancreatic cancer. Our data suggest that GSK-3ß is overexpressed in pancreatic cancer and localizes to the nucleus in the vast majority of moderately and poorly differentiated cancers. Although this study is the first to look at the expression and cellular localization of GSK-3ß in human tumor tissues, several reports have used Western blotting to analyze GSK-3ß protein levels in other cancers. In fact, using Western blotting for GSK-3ß in protein samples from human ovarian cancer specimens, statistical analysis revealed a significant increase of GSK-3ß (P < 0.001) expression in the group of ovarian adenocarcinoma compared with the group of normal ovaries and benign adenomas/borderline tumors (22). In a separate study of human colorectal carcinomas, the level of GSK-3ß protein expression was found to be significantly higher in the tumor tissue than in their normal counterparts (18 of 20 cases), whereas inactive phosphorylated Ser9 GSK-3ß was detected in higher levels in normal tissue than in tumors in 17 of 20 cases (18). Of particular interest, a study using a mouse hepatic carcinogenesis model showed a higher level of GSK-3ß expression in mouse liver tumors than in normal liver tissue as shown by Western blotting (23). However, we are unaware of any previous report that assessed the expression and cellular localization of GSK-3ß in human tumors using immunohistochemistry.
Although GSK-3ß does not contain any identifiable nuclear localization or nuclear export signal sequences, it is known to shuttle from the cytoplasm to the nucleus, where it is thought to participate in the regulation of gene transcription through the phosphorylation of transcription factors (e.g., NFAT and c-Jun; refs. 15, 16). Physiologically, GSK-3ß is expressed in the cytoplasm of normal cells, including pancreatic ductal and acinar cells. However, a significant finding of our study is the presence of nuclear accumulation of GSK-3ß in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. Interestingly, nuclear accumulation of GSK-3ß is significantly correlated with pancreatic cancer dedifferentiation. Another finding of our study is that only the active form of GSK-3ß is detectable in the nucleus of pancreatic cancer cells. Our results clearly show that down-regulation of inactive GSK-3ß in the cancer cell nucleus is independent of its nuclear export. Using the proteasome inhibitor MG132, we could rescue nuclear accumulation of exogenously expressed KD GSK-3ß or pharmacologically inhibited endogenous GSK-3ß. These data suggest that, although inactive GSK-3ß can translocate to the nucleus from the cytoplasm, the inactive form of GSK-3ß is rapidly degraded by the proteasome in the nucleus of the cancer cell. Thus, GSK-3ß kinase activity is required for its enhanced stabilization within the nucleus. However, the exact mechanism by which GSK-3ß becomes overexpressed in human cancers, what regulates its accumulation in the nucleus of pancreatic cancer cells, and what proteasomal pathway is involved in the depletion of inactive GSK-3ß in the nucleus is currently not known. The identification of the signals that drive the aberrant nuclear accumulation of active GSK-3ß in pancreatic cancer cells is currently under investigation in our laboratory.
The exact mechanism by which GSK-3ß affects NF-B activity is currently unknown. Using GSK-3ß-deficient mouse embryonic fibroblasts, it has been shown that the early steps leading to NF-B activation following TNF- treatment (degradation of IB and translocation of NF-B to the nucleus) were unaffected by the loss of GSK-3ß, indicating that NF-B is regulated by GSK-3ß at the level of the transcriptional complex (8). In addition, we have shown recently that GSK-3ß influences NF-B-mediated gene transcription in pancreatic cancer cells at a point downstream of the activation of the IB kinase complex (9). Consistent with the putative role of GSK-3ß in regulating the nuclear activity of NF-B, a recent study showed that loss of GSK-3ß leads to decreased TNF--induced binding of NF-B to the promoters of a subset of target genes, including antiapoptosis genes (e.g., cIAP2), in GSK-3ß-deficient mouse embryonic fibroblasts (24). Of importance, our finding of nuclear accumulation of GSK-3ß suggests the possibility that active GSK-3ß positively regulates NF-B transcriptional activity in the nucleus of pancreatic cancer cells. Although, it is possible that GSK-3ß controls NF-B nuclear activity through direct phosphorylation of NF-B p65, leading to effects on DNA-binding activity or dimerization (25), it is also possible that nuclear GSK-3ß may have an effect on chromatin structure, thereby facilitating accessibility of transcription factors, such as NF-B, at the promoter regions of target genes. Clearly, further studies are required to determine the exact mechanism by which nuclear GSK-3ß can regulate NF-B transcriptional activity.
In summary, our study identifies the overexpression of GSK-3ß in pancreatic cancer and its aberrant nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma. In addition, we have shown that kinase activity contributes to GSK-3ß nuclear accumulation and thereby provides new insight into the mechanism by which GSK-3ß regulates constitutive NF-B activity in pancreatic cancer. Furthermore, we show that inhibition of GSK-3 suppresses pancreatic tumor growth in vivo and affects NF-B-mediated survival and proliferation of cancer cells in established tumor xenografts. Our work suggests that inhibition of GSK-3 is a promising new approach to pancreatic cancer therapy, holding the potential to arrest tumor growth and induce apoptosis in human pancreatic cancer.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
6 A.V. Ougolkov and D.D. Billadeau, unpublished observation. 
Received 1/27/06; revised 5/22/06; accepted 6/23/06.
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B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 1999;5:119–27.B: the enemy within. Cancer Cell 2004;6:203–8.[CrossRef][Medline]B. Nat Med 1999;5:412–7.[CrossRef][Medline]B. Nature 1995;376:167–70.[CrossRef][Medline]B kinase 2 gene. Science 1999;284:321–5.B activation. Nature 2000;406:86–90.[CrossRef][Medline]B-mediated gene transcription and cell survival in pancreatic cancer cells. Cancer Res 2005;65:2076–81.B in colorectal cancer. J Natl Cancer Inst 2004;96:1161–70.}B-Dependent Transcription. Mol Cell Biol 2005;25:8444–55.-induced NF-B activation and apoptosis in hepatocytes. Am J Physiol Gastrointest Liver Physiol 2002;283:G204–11.This article has been cited by other articles:
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  A. V. Ougolkov, N. D. Bone, M. E. Fernandez-Zapico, N. E. Kay, and D. D. Billadeau Inhibition of glycogen synthase kinase-3 activity leads to epigenetic silencing of nuclear factor {kappa}B target genes and induction of apoptosis in chronic lymphocytic leukemia B cells Blood, July 15, 2007; 110(2): 735 - 742. [Abstract] [Full Text] [PDF]
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, diluent (DMSO); 
, AR-A014418. Points, mean relative tumor volume; bars, SE. Table, the number of animals with tumors grown to more than three times the original starting volume in diluent (DMSO) or AR-A014418-treated mice.