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. 2006 Sep 1;12(17):5074-81.
doi: 10.1158/1078-0432.CCR-06-0196.

Aberrant nuclear accumulation of glycogen synthase kinase-3beta in human pancreatic cancer: association with kinase activity and tumor dedifferentiation

Andrei V Ougolkov  1 , Martin E Fernandez-Zapico, Vladimir N Bilim, Thomas C Smyrk, Suresh T Chari, Daniel D Billadeau
Affiliations

Aberrant nuclear accumulation of glycogen synthase kinase-3beta in human pancreatic cancer: association with kinase activity and tumor dedifferentiation

Andrei V Ougolkov et al. Clin Cancer Res. .

Abstract

Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) beta regulates nuclear factor-kappaB (NF-kappaB)-mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3beta 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-3beta affects NF-kappaB activity in pancreatic cancer.

Experimental design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3beta 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-3beta in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3beta nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3beta 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-kappaB-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-3beta nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3beta regulates NF-kappaB activity in pancreatic cancer.

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Figures

Fig. 1
Fig. 1
GSK-3β is overexpressed and accumulated in the nucleus of pancreatic cancer cells. A to E, immunohistochemical analysis of GSK-3β expression and localization in normal human pancreas (A) and pancreatic adenocarcinoma (B–E) specimens. A, normal acinar cells (NAC); normal pancreatic duct (NPD). B, arrows, well-differentiated pancreatic adenocarcinoma shows strong cytoplasmic expression of GSK-3β. C, arrows, nuclear accumulation of GSK-3β found in cancer cells of moderately differentiated adenocarcinoma but not in adjacent PanIN-3 (P3) lesion. Right, higher magnification of the delineated inset (left). D, GSK-3β nuclear accumulation in a moderately differentiated pancreatic adenocarcinoma. E, arrows, nuclear accumulation of GSK-3β was found in cancer cells of poorly differentiated pancreatic adenocarcinoma.
Fig. 2
Fig. 2
Nuclear accumulation of GSK-3β is associated with the loss of pancreatic cancer differentiation. A, distribution of GSK-3β staining patterns in PanIN lesions and pancreatic carcinomas. B to E, immunohistochemical staining of serial sections from the same metastatic lymph node shows accumulation of GSK-3β (B), NF-κB p65 (C), and cyclin D1 (D) in nuclei of cancer cells, whereas β-catenin (E) shows membranous staining.
Fig. 3
Fig. 3
Kinase activity is required for GSK-3β nuclear accumulation in pancreatic cancer cells. A, equivalent amounts (50 μg) of nuclear and cytosolic proteins isolated from the indicated cell lines and normal human pancreas tissue were separated by SDS-PAGE and immunoblotted. B, MIA-PaCa2 cancer cells were transfected with mammalian expression vectors for either HA epitope – tagged (HA.tag) WTor KD GSK-3β. Forty-eight hours after transfection, cells were treated with DMSO (control), leptomycin B (30 ng/mL), or MG132 (10 μmol/L) for 12 hours. Whole-cell lysates (WCL), nuclear (NUC), and cytosolic (CYT) fractions were prepared, separated by SDS-PAGE (50 μg/well), transferred to polyvinylidene difluoride membrane, and probed with the indicated antibodies. Data are representative example of three independent experiments. C, HupT3 pancreatic cancer cells were treated with AR-A014418 (50 μmol/L) for 6, 12, and 24 hours as indicated, nuclear/cytosolic fractions were prepared, and protein expression was analyzed in the two fractions as described in (B). D, CAPAN2 pancreatic cancer cells were treated with AR-A014418 (ARA ; 50 μmol/L), MG132 (10 μmol/L), and combination of these two drugs for 24 hours and nuclear/cytosolic fractions were prepared and analyzed as in (B).
Fig. 4
Fig. 4
Antitumor effect of GSK-3 inhibition in vivo. Established CAPAN2 xenografts (tumor volume, 350-400 mm3) were treated by i.p. injections with DMSO orAR-A014418 (120 mg/kg; every 12 hours for 2 days) and subsequently injected with bromodeoxyuridine (BrDU; 1mg) 3 hours before sacrifice and tumor harvest. A, tumor proteins were extracted from fresh tumor tissues taken from each mouse as indicated and protein expression analysis was done as indicated in Fig. 3. B, TUNEL staining of tissue sections from DMSO- and AR-A014418-treated animals was done to obtain the apoptotic index. Columns, mean; bars, SE. C, proliferation of cancer cells in DMSO-treated and AR-A014418-treated CAPAN2 xenografts was measured by bromodeoxyuridine labeling. Columns, mean; bars, SE. Sections from DMSO-treated (D, F, H, and J) and AR-A014418-treated (E, G, I, and K) CAPAN2 xenograft tumors were stained for histone H3 phosphorylated Ser10 (D and E), cyclin D1 (F and G), XIAP (H and I), or Bcl-2 (J and K). L, CAPAN2 xenograft tumor regrowth assay. Treatment was initiated on day 0 with i.p. injections of either diluent (DMSO) or 30 mg/kg AR-A014418. Arrows, animals were injected daily, five times weekly for 2 weeks.▪, 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) orAR-A014418-treated mice.
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