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Tgfb1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions Brook S

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Tgfb1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions Brook S Published OnlineFirst September 26, 2018; DOI: 10.1158/1541-7786.MCR-18-0558 Signal Transduction Molecular Cancer Research TGFb1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-deficient Conditions Brook S. Nepon-Sixt and Mark G. Alexandrow Abstract Transforming growth factor b1 (TGFb1) is a potent inhib- expression prevents TGFb1 suppression of MCM assembly. itor of cell growth that targets gene-regulatory events, but Mechanistically, TGFb1 blocks a Cyclin E–Mcm7 molecular also inhibits the function of CDC45-MCM-GINS helicases interaction required for MCM hexamer assembly upstream of (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go- CDC10-dependent transcript-1 (CDT1) function. Accordingly, Ichi-Ni-San) through multiple mechanisms to achieve cell- overexpression of CDT1 with an intact MCM-binding domain cycle arrest. Early in G1, TGFb1 blocks MCM subunit expres- abrogates TGFb1 arrest and rescues MCM assembly. The ability sion and suppresses Myc and Cyclin E/Cdk2 activity required of CDT1 to restore MCM assembly and allow S-phase entry for CMG assembly, should MCMs be expressed. Once CMGs indicates that, in the absence of Rb and other canonical med- are assembled in late-G1, TGFb1 blocks CMG activation using iators, TGFb1 relies on inhibition of Cyclin E-MCM7 and MCM a direct mechanism involving the retinoblastoma (Rb) tumor assembly to achieve cell cycle arrest. suppressor. Here, in cells lacking Rb, TGFb1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth Implication: These results demonstrate that the MCM assem- arrest remains intact and Smad2/3/4-dependent. Such arrest bly process is a pivotal target of TGFb1 in eliciting cell cycle occurs due to inhibition of MCM hexamer assembly by TGFb1, arrest, and provide evidence for a novel oncogenic role for which is not seen when Rb is present and MCM subunit CDT1 in abrogating TGFb1 inhibition of MCM assembly. expression is normally blocked by TGFb1. Loss of Smad Mol Cancer Res; 1–12. Ó2018 AACR. Introduction occur in cells lacking p27Kip1, p15INK4B, or p21Cip1 (13–16), and downregulation of c-Myc is not always associated with The ability of TGFb1 to inhibit cell proliferation is often lost TGFb1-induced arrest (17). Perhaps most significant, TGFb1 can in cancers due to abrogation of the molecular events mediating block cell cycle progression in early-G1 in cells that are lacking TGFb1 control over the cell cycle. TGFb1 cell cycle arrest occurs À À the Rb protein (18) or in Rb / fibroblasts under low plating during G phase and is governed by Smad signaling proteins 1 conditions (19). Such studies suggest that other unknown that elicit multiple negative effects on the cell cycle machinery mechanisms exist that mediate TGFb1 arrest and are independent via transcriptional regulation (1, 2). Expression of c-Myc is of c-Myc suppression, kinase inhibition, and Rb function. acutely downregulated (3), which allows induction of the Insight into these questions derives from a role for TGFb1 cyclin-dependent kinase inhibitor (CKI) p15INK4B (4–6). This growth-inhibitory signals in suppressing Mini-Chromosome and other CKIs, including p21Cip1 and p27Kip1, are utilized Maintenance (MCM) complex assembly and activation (18). The by TGFb1 to inhibit the activity of Cyclin D/Cdk4 and Cyclin MCM complex is a heterohexamer comprised of Mcm2-7 subunits E/Cdk2 kinases (7–10). The suppression of these kinases pre- and represents the core of the replicative DNA helicase (20). The vents the hyperphosphorylation of the retinoblastoma protein MCM complex is recruited to chromatin as part of the pre- (Rb), resulting in the maintenance of Rb in a hypophosphory- Replication Complex (pre-RC) that regulates initiation of DNA lated and growth-inhibitory form that inhibits progression into replication from chromosomal origins (21). In synchronized S-phase (11, 12). mammalian cells, MCMs are recruited in late-G (18, 22). The An important feature of the canonical growth-inhibitory sig- 1 DNA binding of MCM hexamers depends on the combined naling pathway for TGFb1 is that the growth-suppressive form of functions of Cdc6 (cell division cycle complementation group 6) Rb is thought to play a pivotal role in the cell cycle arrest that and Cdt1 (Cdc10-dependent transcript-1) proteins (23), and occurs (11, 12). However, several studies suggest that Rb, c-Myc prior to the G –S transition Cdc45 and GINS (Go-Ichi-Ni-San, suppression, and kinase inhibition may be dispensable for 1 Japanese for 5-1-2-3 defining the protein subunits Sld5 and TGFb1-induced growth arrest. Growth inhibition by TGFb1 can Psf1,2,3) are recruited to the prebound MCM hexamers, forming replicative CMG (Cdc45-MCM-GINS) helicases (24). Treatment of cells with TGFb1 in early-G1 suppresses MCM protein expres- Department of Molecular Oncology, Moffitt Cancer Center and Research sion, thereby precluding hexamer assembly on chromatin because Institute, Tampa, Florida. MCMs are absent (18). This contributes to growth arrest alongside Corresponding Author: Mark G. Alexandrow, Moffitt Cancer Center, c-Myc suppression and kinase inhibition. However, once MCMs MRC-4072E, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-1450; are loaded in late-G1, TGFb1 can still arrest cell cycle progression Fax: 813-745-6180; E-mail: Mark.Alexandrow@Moffitt.org by inhibiting activation of assembled CMG helicases (18). The doi: 10.1158/1541-7786.MCR-18-0558 latter requires an interaction of Rb with the helicases in late-G1 Ó2018 American Association for Cancer Research. that inhibits their function via an N-terminal domain of Rb lost in www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 26, 2018; DOI: 10.1158/1541-7786.MCR-18-0558 Nepon-Sixt and Alexandrow cancers (18, 25). Upon loss of Rb, TGFb1 is rendered ineffective in trogen:); anti-Rb (#PC13; 1:500; Calbiochem); rabbit monoclo- late-G1 (18). nal anti-Smad4 (#04-1033; 1:750; Millipore); mAb anti-Mcm6 The ability of TGFb1toefficiently arrest cells in early-G1 (#sc-55577; 1:200), mAb anti-Mcm7 (#sc-9966; 1:1,000), anti- when Rb is absent is unexpected (18). Remarkably, such Cdc6 (#sc-8341; 1:300), anti-Cdc6-serine54P (#sc-12920R; TGFb1 inhibition also occurs in the absence of suppressive 1:500), anti-CycE (agarose-conjugated; #sc-481; 1:20), mAb effects on MCM expression, c-Myc, or Cyclin E/CDK2 kinase anti-Cyclin E (#sc-377100; 1:100), and anti-Cdt1 (#sc-28262; activity. We describe here a previously unknown mechanism 1:300; Santa Cruz Biotechnology); mAb anti-actin (#A5316; used by TGFb1toblockG1 progression when Rb and these 1:5,000; from Sigma); anti-Mcm2 (1:1,000) was generated by canonical mediators of TGFb1 inhibitory signals are not our group and validated previously (27); anti-Mcm5 (1:2000) required for the growth arrest. TGFb1 elicits arrest by directly was provided by Rolf Knippers (University of Konstanz, Konstanz, disrupting the recruitment and loading of MCM hexamers Germany); anti-Myc (1:3,000) was provided by Steve Hann onto DNA. TGFb1achievesthisbysuppressingaCyclin (Vanderbilt University, Nashville, TN); anti-HBO1 (1:200) was E–MCM interaction that is required for the Cdt1-directed loading provided by Masayoshi Iizuka (Teikyo University School of of MCMs (26). Accordingly, overexpression of the Cdt1 oncopro- Medicine, Tokyo, Japan). tein in Rb-deficient cells abrogates TGFb1-induced growth arrest, restores MCM loading, and allows G1–S transit. These results Immunofluorescence provide evidence for a novel oncogenic function for Cdt1 in Replicating DNA was labeled by pulsing for 30 minutes with overcoming TGFb1 signals. Furthermore, these findings demon- 15 mmol/L bromodeoxyuridine (BrdU) prior to fixation. BrdU strate that there is additional redundancy in the mechanisms detection and immunofluorescence (IF) assays were performed underlying TGFb1 growth arrest, and highlight how cancer cells using standard procedures. For detection of chromatin-bound can overcome such normal growth-regulatory signals that control Mcm2 by IF, prior to fixation cells were preextracted for 5 minutes cell cycle progression. with cold extraction buffer [0.1% Triton-X100, phosphate- buffered saline pH 7.4, 5 mmol/L MgCl2, 0.1 mmol/L ethylene- diamine tetraacetic acid (EDTA) pH 8]. For Mcm2 IF loading Materials and Methods assays, the same buffer was used, but included 80 mmol/L KCl Cell culture, synchronization, and transfections (high salt). Mouse keratinocytes (Balb/MK) were obtained from the Har- old Moses laboratory, assessed for lack of mycoplasma and other Immunoprecipitations, kinase assays, and immunoblotting bacterial contaminants, and maintained as described (18). MK Immunoprecipitations (IP) were performed at 4C on equal cells (nonprimary) are routinely tested for TGFb1 sensitivity and numbers of cells lysed in TNN buffer [50 mmol/L Tris-HCl pH 8, synchronization efficacy, and cultured no longer than 6 weeks in 250 mmol/L NaCl, 0.1% Nonidet P-40, 5 mg/mL Aprotinin, low calcium MEM to maintain proper characteristics and prevent 5 mg/mL Leupeptin, 1 mmol/L phenylmethanesulfonyl fluoride terminal differentiation. TGFb1 (recombinant human; R&D Sys- (PMSF)]. Samples were precleared with secondary beads for 30 tems) was used at 10 ng/mL. Synchronization was achieved by minutes. Primary antibodies or rabbit IgG were rocked with culturing MK cells in medium lacking EGF for 3.5 days. Transfec- lysates for 2 hours, then 1 hour with anti-rabbit agarose beads tions utilized Fugene6 (Roche) for plasmids, and DharmaFECT-4 (Sigma), followed by 4 Â 15-minute (rocking) bead washes in (Dharmacon) for siRNA. Cdt1 mutants were generated using excess TNN buffer. For immunoprecipitation–Western blot assays standard proof-reading (Pfu-polymerase-based) PCR methods. with anti-Cyclin E (final 8 mg/mL), anti-Cyclin E was conjugated Luciferase or Smad siGENOME SMARTpool siRNA (Dharmacon) to agarose beads without the need for the secondary incubation were transfected following EGF removal (25 nmol/L final con- step.
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