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Role of Glycogen Synthase Kinase 3B in Rapamycin-Mediated Cell Cycle

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Role of Glycogen Synthase Kinase 3B in Rapamycin-Mediated Cell Cycle Research Article Role of Glycogen Synthase Kinase 3 B in Rapamycin-Mediated Cell Cycle Regulation and Chemosensitivity JinJiang Dong,1 Junying Peng,1 Haixia Zhang,1 Wallace H. Mondesire,1 Weiguo Jian,1 Gordon B. Mills,2 Mien-Chie Hung,1,3 and Funda Meric-Bernstam1 Departments of 1Surgical Oncology, 2Molecular Therapeutics, and 3Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas Abstract Introduction The mammalian target of rapamycin is a serine-threonine Rapamycin and its analogues are being actively investigated in kinase that regulates cell cycle progression. Rapamycin and clinical trials as novel targeted anticancer agents. The mammalian its analogues inhibit the mammalian target of rapamycin target of rapamycin (mTOR) is a serine-threonine kinase that and are being actively investigated in clinical trials as novel regulates cell cycle progression. The two best-studied targets of targeted anticancer agents. Although cyclin D1 is down- mTOR, eukaryotic initiation factor 4E-binding protein 1 and regulated by rapamycin, the role of this down-regulation in ribosomal p70 S6 kinase-1, are thought to modulate translation; rapamycin-mediated growth inhibition and the mechanism thus, rapamycin is thought to alter the translation of mRNA of cyclin D1 down-regulation are not well understood. Here, involved in control of the cell cycle. However, how rapamycin we show that overexpression of cyclin D1 partially over- blocks cell growth and proliferation is not well understood. comes rapamycin-induced cell cycle arrest and inhibition of Prior studies have suggested that cyclin D1 is a key target of anchorage-dependent growth in breast cancer cells. Rapa- mTOR (1–4). Cyclin D1 is overexpressed in a variety of tumor mycin not only decreases endogenous cyclin D1 levels but types and is proposed to contribute to cancer development. also decreases the expression of transfected cyclin D1, Cyclin D1 plays a critical role in G progression by activating suggesting that this is at least in part caused by accelerated 1 cyclin-dependent kinases 4 and 6, leading to phosphorylation of proteolysis. Indeed, rapamycin decreases the half-life of tumor suppressor pRb, with depression of E2F-mediated cyclin D1 protein, and the rapamycin-induced decrease in transcription (5). Independent of cyclin-dependent kinase cyclin D1 levels is partially abrogated by proteasome activity, cyclin D1 also modulates other transcription factors, inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treat- such as estrogen and androgen receptors, signal transducers and ment leads to an increase in the kinase activity of glycogen activators of transcription 3, and PPARg (6, 7). Recently, it has synthase kinase 3B (GSK3B), a known regulator of cyclin D1 been reported that transcription factor CCAAT/enhancer-binding proteolysis. Rapamycin-induced down-regulation of cyclin protein h is involved in regulating genes affected by cyclin D1 D1 is inhibited by the GSK3B inhibitors lithium chloride, overexpression (8). Although the exact role of cyclin D1 SB216763, and SB415286. Rapamycin-induced G arrest is 1 overexpression in human tumors is controversial, there are abrogated by nonspecific GSK3B inhibitor lithium chloride several lines of evidence indicating that cyclin D1 plays a crucial but not by selective inhibitor SB216763, suggesting that role in mammary gland carcinogenesis. First, cyclin D1 is GSK3B is not essential for rapamycin-mediated G arrest. 1 overexpressed in ductal carcinoma in situ and invasive ductal However, rapamycin inhibits cell growth significantly more in breast carcinoma and cyclin D1 overexpression is associated GSK3B wild-type cells than in GSK3B-null cells, suggesting with a poorer prognosis (9, 10). Second, mammary gland– that GSK3B enhances rapamycin-mediated growth inhibition. targeted cyclin D1 overexpression in mouse mammary tumor In addition, rapamycin enhances paclitaxel-induced apopto- virus-cyclin D1 transgenic mice leads to mammary hyperplasia sis through the mitochondrial death pathway; this is and development of carcinoma, suggesting that cyclin D1 at inhibited by selective GSK3B inhibitors SB216763 and least is a weak oncogene (11). Third, mice lacking cyclin D1 are SB415286. Furthermore, rapamycin significantly enhances resistant to mammary carcinogenesis by the H-ras and HER-2/ paclitaxel-induced cytotoxicity in GSK3B wild-type but not in neu oncogenes, suggesting that cyclin D1 expression is critical to GSK3B-null cells, suggesting a critical role for GSK3B in H-ras and HER-2/neu-mediated carcinogenesis (12). Finally, rapamycin-mediated paclitaxel-sensitization. Taken together, B deregulation of cyclin D1 is implicated as the central pathway these results show that GSK3 plays an important role in involved in chemical carcinogenesis models of breast cancer rapamycin-mediated cell cycle regulation and chemosensi- (13). Therefore, cyclin D1 may represent an important down- tivity and thus significantly potentiates the antitumor effects stream target of signaling pathways that have a role in of rapamycin. (Cancer Res 2005; 65(5): 1961-72) mammary carcinogenesis, making the effect of rapamycin on cyclin D1 of particular interest. Cyclin D1 expression is regulated at multiple levels. Cyclin D1 transcription is up-regulated by mitogen stimulation through extracellular signal-regulated kinase 1/2 and 5, c-Jun NH -terminal Note: J. Dong and J. Peng contributed equally to this work. 2 Requests for reprints: Funda Meric-Bernstam, Department of Surgical kinase, signal transducers and activators of transcription 5, nuclear Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe factor-nB, and h-catenin and down-regulated by stress-activated Boulevard, Unit 444, Houston TX 77030. Phone: 713-745-4453; Fax: 713-745-4926; E-mail: [email protected]. kinase p38 (14–16). Cyclin D1 mRNA stability is regulated by a I2005 American Association for Cancer Research. sequence in its 3V untranslated region; its stability is increased by www.aacrjournals.org 1961 Cancer Res 2005; 65: (5). March 1, 2005 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Cancer Research the phosphatidylinositol 3-kinase (PI3K) pathway and is decreased Colonies of >120 um in diameter (20 cells) were counted using a by prostaglandin A (14, 17). Cyclin D1 mRNA translation is thought microscope. to be controlled by the PI3K/Akt pathway and by mTOR signaling Cell Cycle Analysis. Cells were trypsinized and washed with PBS, and (18). Cyclin D1 protein stability is regulated by glycogen synthase cells used in GFP experiments were fixed with 0.5% paraformaldehyde for 10 minutes. These cells were washed with PBS and then fixed with 75% ethanol kinase 3h (GSK3h) and p38, which phosphorylate the protein, overnight at 4jC. Following this, the cells were then washed twice with PBS triggering ubiquitination (19, 20). Cyclin D1 mRNA translation is and resuspended in hypotonic propidium iodide solution (10 Ag propidium often called the critical mode of cyclin D1 regulation by rapamycin. iodide, 10 Ag RNase A, 0.5% Tween 20 in 1 mL PBS) for 0.5 hour at 37jC and In recent work, Gera et al. have shown that rapamycin leads to a kept in the dark at 4jC before analysis. Cell cycle distribution was 5-fold decrease in cyclin D1 translation in cell lines that have high determined with a FACScan flow cytometer and Cell Quest software levels of Akt activity (3). However, in NIH3T3 cells, rapamycin was (Becton Dickinson, San Jose, CA). also found to decrease cyclin D1 mRNA and protein stability Western Blot Analysis. Cultured cells were washed with cold PBS and through unknown mechanisms (21). Therefore, the mechanism by lysed in radioimmunoprecipitation buffer [20 mmol/L Tris (pH 7.5), 150 which rapamycin down-regulates cyclin D1 expression needs mmol/L NaCl, 5 mmol/L EDTA, 1% NP40, 1 mmol/L Na3VO4, 1 mmol/L further study. phenylmethylsulfonyl fluoride, 50 mmol/L NaF] supplemented with complete protease inhibitors on ice. To extract cytosolic proteins for In our previous work, we found that rapamycin leads to down- detection of cytochrome c release, 4 Â 106 cells were harvested and regulation of cyclin D1 levels in rapamycin-sensitive breast cancer washed twice with ice-cold PBS and resuspended in 300 AL ice-cold buffer cell lines MCF-7 and MDA-MB-468 but not in rapamycin-resistant [20 mmol/L HEPES-KOH (pH 7.0), 10 mmol/L KCl, 1.5 mmol/L MgCl2, cell lines MDA-MB-231, MDA-MB-435, and NCI/ADR-RES, suggesting 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 250 mmol/L sucrose, that rapamycin-mediated cyclin D1 down-regulation may be 1 Ag/mL leupeptin and pepstatin, 2 Ag/mL aprotinin]. After incubation on critical to its growth-inhibitory effect (22). In this study, we found ice for 15 minutes, cells were homogenized with a Dounce homogenizer that cyclin D1 plays an important role in rapamycin-mediated cell (B pestle per 25 strokes) and centrifuged at 1,000 Â g for 10 minutes. The cycle arrest and growth inhibition. We show that rapamycin supernatants were centrifuged at 14,000 Â g for 15 minutes in a mediates cyclin D1 down-regulation in part by accelerated microcentrifuge to pellet membranes, including mitochondria. The proteolysis. Furthermore, we show that rapamycin activates resulting supernatants were used as cytosolic extracts. Cell lysates or cytosolic extracts were separated by SDS-PAGE and transferred to a 0.2 Am GSK3h and that GSK3h activity is critical for rapamycin-mediated h polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). cyclin D1 down-regulation. We show that although GSK3 is not Membranes were blocked with 5% nonfat dry milk in TBST (TBS with essential for rapamycin-mediated growth inhibition, it significantly 0.1% Tween 20) and immunoblotted with antibodies. The immunoblots enhances the growth-inhibitory effect of rapamycin. Further, were visualized by an enhanced chemiluminescence detection system rapamycin-mediated chemosensitization is also attenuated in (Amersham Life Sciences, Arlington Heights, IL).
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