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Deregulated Gsk3b Activity in Colorectal Cancer: Its Association with Tumor Cell Survival and Proliferation Q BBRC Biochemical and Biophysical Research Communications 334 (2005) 1365–1373 www.elsevier.com/locate/ybbrc Deregulated GSK3b activity in colorectal cancer: Its association with tumor cell survival and proliferation q Abbas Shakoori a,b,1, Andrei Ougolkov a,d,1, Zhi Wei Yu a, Bin Zhang a, Mohammad H. Modarressi c, Daniel D. Billadeau d, Masayoshi Mai a, Yutaka Takahashi a, Toshinari Minamoto a,b,* a Divisions of Diagnostic Molecular Oncology and Surgical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan b Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan c Department of Medical Genetics, Tehran University of Medical Sciences, Keshavarz Blvd, Tehran, Iran d Division of Oncology Research, Department of Immunology, Mayo Clinic College of Medicine, MN 55905, USA Received 27 June 2005 Available online 19 July 2005 Abstract Glycogen synthase kinase 3b (GSK3b) reportedly has opposing roles, repressing Wnt/b-catenin signaling on the one hand but maintaining cell survival and proliferation through the NF-jB pathway on the other. The present investigation was undertaken to clarify the roles of GSK3b in human cancer. In colon cancer cell lines and colorectal cancer patients, levels of GSK3b expres- sion and amounts of its active form were higher in tumor cells than in their normal counterparts; these findings were indepen- dent of nuclear accumulation of b-catenin oncoprotein in the tumor cells. Inhibition of GSK3b activity by phosphorylation was defective in colorectal cancers but preserved in non-neoplastic cells and tissues. Strikingly, inhibition of GSK3b activity by chem- ical inhibitors and its expression by RNA interference targeting GSK3b induced apoptosis and attenuated proliferation of colon cancer cells in vitro. Our findings demonstrate an unrecognized role of GSK3b in tumor cell survival and proliferation other than its predicted role as a tumor suppressor, and warrant proposing this kinase as a potential therapeutic target in colorectal cancer. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Glycogen synthase kinase 3b; Phosphorylation; Colorectal cancer; Cell survival; Proliferation; Apoptosis; Therapeutic target; Wnt signaling; b-catenin; NF-jB Activation of the Wnt/b-catenin signaling pathway human colorectal cancer and demonstrated that activa- has emerged as an oncogenic pathway leading to colo- tion of this oncoprotein in the tumor invasion front rectal cancer development [1,2]. In an earlier study, we reliably identifies a subset of colon cancer patients identified distinct patterns of b-catenin activation in who are susceptible to tumor recurrence and have a less favorable survival rate [3]. These clinical observations q Abbreviations: APC, adenomatous polyposis coli; CTNNB1, establish the importance of molecular mechanism(s) b-catenin gene; GSK3b, glycogen synthase kinase 3b; NF-jB, nuclear underlying the distinct patterns of b-catenin activation. factor-jB; NSAID(s), non-steroidal anti-inflammatory drug(s); NID- Among regulators of the Wnt signaling pathway [2] is DM, non-insulin-dependent diabetes mellitus; PKB, protein kinase B; glycogen synthase kinase 3b (GSK3b), which phosphor- RNAi, RNA interference; siRNA, small interfering RNA. * Corresponding author. Fax: +81 76 234 4523. ylates b-catenin, thereby recruiting it for ubiquitin-med- E-mail address: [email protected] (T. Minamoto). iated degradation under physiological conditions [2,4,5]. 1 These authors contributed equally to this study. Since GSK3b is a negative regulator of Wnt/b-catenin 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.041 1366 A. Shakoori et al. / Biochemical and Biophysical Research Communications 334 (2005) 1365–1373 signaling, clarifying whether GSK3b plays a part in the Materials and methods mechanism underlying the different patterns of oncogenic b-catenin activation is a matter of consider- Cell lines. Colon cancer cell lines (SW480, SW620, HT29, LoVo, able interest. HCT116, SW48, and RKO) and HEK293 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells of GSK3b is a multifunctional serine/threonine kinase each type were grown and harvested for extraction of protein and that regulates various cellular pathways, depending on DNA. Genetic alterations in all but one of the colon cancer cell lines its substrates for phosphorylation [4–6], and it is evident have been documented (Table 1) [12]. that regulation of Wnt/b-catenin signaling is only one of Patients and tissue samples. Subjects comprised 20 patients who its diverse functions [4,5]. Since oncogenic transcription underwent surgical removal of colorectal cancer in our institute in the period 2002–2003. Clinical data, tumor stage at initial treatment factors (e.g., c-Jun, c-Myc) and proto-oncoproteins (i.e., according to the TNM classification [13], and presence of distant b-catenin, Gli proteins) are putative GSK3b substrates metastasis and recurrence are shown in Table 2. All patients agreed to for phosphorylation-dependent inactivation [4],itis enrollment in the study and each gave informed consent. The Institu- hypothesized that GSK3b interferes with cellular neo- tional Review Board of Kanazawa University approved all protocols plastic transformation and tumor development, as exem- upon patientsÕ agreement. A pair of normal and tumor tissue samples was obtained from each fresh surgical specimen and stored at À80 °C plified by its activity in Wnt/b-catenin signaling [2]. until use. However, only a few studies have addressed its role(s) in In the light of evidence that nuclear accumulation (NA) of b-ca- human cancer, and these studies have reported differing tenin is an indicator of oncogenic activity [2], b-catenin activation in effects of GSK3b on cancer cells [7,8]. Using GSK3b defi- the primary tumor was determined by immunohistochemistry using the cient mouse embryonic fibroblasts, it was shown that antibody to b-catenin (BD Biosciences) as described in our previous study [3]. Each instance of b-catenin expression in the primary tumor GSK3b plays a crucial role in cell survival mediated by was classified into one of three distinct previously defined patterns [3]: the nuclear factor-jB (NF-jB) pathway [9,10]. Interest- membranous expression (M), imitating that found in normal crypts; ingly, we have previously shown that the Wnt/b-catenin diffuse NA (NAd), defined as carcinoma cells with b-catenin-positive and NF-jB pathways were co-activated in colorectal can- nuclei distributed throughout the tumor; and NA only in the tumor cer by dysregulation in the ubiquitin system [11]. Thus, invasion front (NAinv). Western blotting analysis. Cellular protein was extracted from frozen these observations bring forward apparently opposing surgical specimens and cell lines using lysis buffer (CelLytic-MT, Sigma– notions regarding the functions of GSK3b in neoplastic Aldrich, St. Louis, MO) in a mixture of protease and phosphatase cells on the one hand, removing a neoplastic trigger by inhibitors (both from Sigma–Aldrich). A 100 lg aliquot of protein ex- phosphorylation-dependent degradation of b-catenin tract was subjected to Western blotting analysis as described previously oncoprotein in the ubiquitin system, and on the other, [11] to examine expression of the respective kinases using primary anti- bodies against GSK3b (diluted 1:2,500; BD Biosciences, Lexington, KY) contributing to a cell proliferation and survival pathways and its fractions phosphorylated at the serine 9 residue (phospho- by regulating NF-jB. GSK3bSer9) (diluted 1:1,000; Cell Signaling Technology, Beverly, MA), The present study was therefore undertaken to clarify and the tyrosine 216 (phospho-GSK3bTyr216) (diluted 1:1,000; BD Bio- the role of GSK3b in cancer by analyzing expression sciences), respectively. As a representative of activated kinases upstream and activity of this kinase in colon cancer cell lines and to GSK3b, protein kinase B (PKB, also termed Akt) activity was determined by immunoblotting with antibodies to phospho-AktThr308 clinical colorectal cancers and investigating its effects on and phospho-AktSer473 (both diluted 1:1,000) (Cell Signaling Technol- cancer cells. Our results demonstrate that independently ogy). In each case, signals were developed using an enhanced chemilu- of its effects on Wnt/b-catenin signaling, GSK3b has a minescent detection reagent (ECL, Amersham, Little Chalfont, UK). hitherto unrecognized pathologic role in cell survival In vitro kinase assay. GSK3b was immunoprecipitated from the cell and proliferation in colorectal cancer, and suggest the lines and subjected to an in vitro kinase assay using a recombinant human b-catenin protein (generated in our laboratory) as a substrate necessity of further studies to evaluate GSK3b as a poten- according to the method described previously [14]. The phosphorylated tial therapeutic target for cancer treatment. fraction of b-catenin was detected by autoradiography. Table 1 Comparison of colon cancer cell lines in regard to GSK3b phosphorylation and mutations in APC, CTNNB1, and K-ras Cell lines Mutation [12] Phosphorylation APC CTNNB1a K-rasb GSK3bSer9 GSK3bTyr216 AktTyr308 AktSer473 SW480 + À 12 À +++ SW620 + À 12 À ++À HT29 + ÀÀÀ ++À LoVo + À 13 À +++ HCT116 À 45 13 À ++À SW48 À 33 ÀÀ + Trace À RKO NA ÀÀÀ + Trace + +, present; À, absent or undetectable; NA, data not available. a 45, 33: mutation in codon 45 or 33. b 12, 13: mutation in codon
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