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LETTER Doi:10.1038/Nature09779 LETTER doi:10.1038/nature09779 Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7 Ingrid E. Wertz1, Saritha Kusam1, Cynthia Lam1*, Toru Okamoto2*, Wendy Sandoval3, Daniel J. Anderson4, Elizabeth Helgason1, James A. Ernst1,3, Mike Eby4, Jinfeng Liu5, Lisa D. Belmont4, Joshua S. Kaminker5, Karen M. O’Rourke6, Kanan Pujara7, Pawan Bir Kohli8, Adam R. Johnson8, Mark L. Chiu9, Jennie R. Lill3, Peter K. Jackson4, Wayne J. Fairbrother1, Somasekar Seshagiri7, Mary J. C. Ludlam4, Kevin G. Leong4, Erin C. Dueber1, Heather Maecker4, David C. S. Huang2,10 & Vishva M. Dixit6 Microtubules have pivotal roles in fundamental cellular processes (IAP) proteins5 do not have a significant role in the cellular response to and are targets of antitubulin chemotherapeutics1. Microtubule- antitubulin agents (Supplementary Fig. 3), we conclude that BCL2 targeted agents such as Taxol and vincristine are prescribed widely family proteins are key regulators of antitubulin-agent-induced cell for various malignancies, including ovarian and breast adenocarci- death in diverse cell types. nomas, non-small-cell lung cancer, leukaemias and lymphomas1. Next we determined the sensitivity of MEFs lacking individual BCL2 These agents arrest cells in mitosis and subsequently induce cell family members to killing by Taxol or vincristine, two mechanistically death through poorly defined mechanisms2. The strategies that res- distinct antitubulin chemotherapeutics. Bclx2/2 cells were more sensi- istant tumour cells use to evade death induced by antitubulin agents tive to Taxol than were WT cells, and Mcl12/2 cells showed greater are also unclear2. Here we show that the pro-survival protein MCL1 sensitivity than WT cells to Taxol or vincristine (Fig. 1c, d). Because the (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin ratio of pro-survival to pro-apoptotic BCL2 family proteins dictates cell chemotherapeutics. During mitotic arrest, MCL1 protein levels fate3, we monitored the levels of these proteins during mitotic arrest, as decline markedly, through a post-translational mechanism, poten- indicated by phosphorylation of the anaphase-promoting complex tiating cell death. Phosphorylation of MCL1 directs its interaction subunit CDC27 (ref. 6). MCL1 protein levels declined markedly in with the tumour-suppressor protein FBW7, which is the substrate- synchronized cells released into nocodazole or Taxol (Fig. 1e and Sup- binding component of a ubiquitin ligase complex. The polyubiqui- plementary Fig. 4). The decrease in NOXA protein levels is probably tylation of MCL1 then targets it for proteasomal degradation. The an indirect consequence of MCL1-regulated stability (D.C.S.H., unpub- degradation of MCL1 was blocked in patient-derived tumour cells lished observations). MCL1 protein levels also declined in unsynchro- that lacked FBW7 or had loss-of-function mutations in FBW7, nized cells that were arrested in mitosis (Supplementary Figs 5 and 34). conferring resistance to antitubulin agents and promoting chemo- MCL1 transcription was not significantly decreased during mitotic therapeutic-induced polyploidy. Additionally, primary tumour arrest in human cell lines (Fig. 2a). This implicated a role for the samples were enriched for FBW7 inactivation and elevated MCL1 ubiquitin–proteasome system, the primary conduit for regulated pro- levels, underscoring the prominent roles of these proteins in onco- tein degradation in eukaryotic cells7, in the reduction of MCL1 protein genesis. Our findings suggest that profiling the FBW7 and MCL1 levels. Indeed, the proteasome inhibitor MG132 blocked MCL1 degra- status of tumours, in terms of protein levels, messenger RNA levels dation (Fig. 2b and Supplementary Fig. 6), and endogenous MCL1 was and genetic status, could be useful to predict the response of patients ubiquitylated during mitotic arrest (Supplementary Fig. 7). to antitubulin chemotherapeutics. MCL1 contains potential degron motifs for association with the BCL2 family proteins are key regulators of cell survival and can either F-box proteins b-transducin-repeat-containing protein (b-TRCP; also promote or inhibit cell death3. Pro-survival members, including BCL- known as FBXW1 or FWD1)8 and FBW7 (also known as FBXW7, 9 XL and MCL1, inhibit apoptosis by blocking the cell death mediators AGO, CDC4 or SEL10) (Supplementary Fig. 8). F-box proteins are BAX and BAK (also known as BAK1). When uninhibited, BAX and substrate receptors for SKP1–CUL1–F-box (SCF)-type ubiquitin ligase BAK permeabilize the outer mitochondrial membranes, which releases complexes, which mediate degradative polyubiquitylation9,10. Con- pro-apoptotic factors that activate caspases, the proteases that catalyse sistent with a role for CUL1-based ubiquitin ligases in MCL1 turnover, cellular demise. This intrinsic, or mitochondrial, pathway is initiated by ectopic expression of a dominant-negative CUL1 protein blocked the damage-sensing BH3-only proteins, including BIM (encoded by MCL1 degradation during mitotic arrest (Supplementary Fig. 9). BCL2L11) and NOXA (also known as PMAIP1), which neutralize the These data indicate that CUL1-containing ubiquitin-ligase complexes pro-survival family members when cells are irreparably damaged4. have a more prominent role in regulating MCL1 turnover during Because aberrant expression of pro-survival BCL2 family proteins mitotic arrest than MULE, a ligase that ubiquitylates MCL1 (ref. 11), promotes tumorigenesis and resistance to chemotherapeutics3,we an idea corroborated by knocking down MULE expression in Taxol- evaluated whether these proteins regulate the cell death induced by treated cells by using RNA interference (RNAi) (Supplementary Fig. antitubulin agents. Multiple lineages of Bax2/2Bak2/2 mouse embry- 10a–c). RNAi-mediated knockdown of FBW7 expression, but not onic fibroblasts (MEFs) were resistant to killing by Taxol or nocoda- b-TRCP expression, attenuated MCL1 degradation in tumour cells zole, whereas wild-type (WT) MEFs were significantly more sensitive (Fig. 2c and Supplementary Figs 11 and 12) and untransformed cells to such killing (Fig. 1a and Supplementary Fig. 2a–e). These results (Supplementary Fig. 13a, b). MCL1 degradation (Fig. 2d) and turnover were confirmed in myeloid cells (Fig. 1b). As inhibitor of apoptosis (Supplementary Fig. 14) was protracted in FBW7-null cells relative to 1Department of Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, USA. 2The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. 3Department of Protein Chemistry, Genentech, South San Francisco, California 94080, USA. 4Department of Research Oncology, Genentech, South San Francisco, California 94080, USA. 5Department of Bioinformatics, Genentech, South San Francisco, California 94080, USA. 6Department of Physiological Chemistry, Genentech, South San Francisco, California 94080, USA. 7Department of Molecular Biology, Genentech, South San Francisco, California 94080, USA. 8Department of Biochemical Pharmacology, Genentech, South San Francisco, California 94080, USA. 9Department of Structural Biology, Abbott Laboratories, Abbott Park, Illinois 60064, USA. 10Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia. *These authors contributed equally to this work. 110 | NATURE | VOL 471 | 3 MARCH 2011 ©2011 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Bax–/–Bak–/– 150 HCT 116 cells 150 HeLa cells ab clone 1-4 a b – MG132 + MG132 Bax–/–Bak–/– clone 2-5A 120 120 Bax–/–Bak–/– Mitotic time clone 6-2 100 100 course (h): –6–4–2 M +3+6 +9 –6–4–2 M +3 +6 +9 Bax–/–Bak–/– E1A/RAS WT clone 4-1 80 80 WB: Tubulin Tubulin 50- MCL1 120 E1A/RAS WT 120 WT clone 4-2 +MCL1 mRNA (%) 60 60 WT clone 4-8 WB: CDC27 CDC27-P 40 40 CDC27 100 100 98- MCL1 20 20 80 80 Mitotic time 0 0 course (h): –4 –2 M +3 +6 +9 –4 –2 M +3 +6 +9 Tubulin 60 60 WB: Tubulin50- 50- + MCL1 MCL1 CDC27-P Viability (%) 40 Viability (%) 40 WB: CDC27 98- 98- CDC27 20 20 c RNAi oligo: BTRC-specific FBW7-specific Scrambled Mitotic time 0 0 course (h): –8 –2 M +2 +4 +6 +8 –8 –2 M +2 +4 +6 +8 –8 –2 M +2 +4 +6 +8 0 200 400 600 800 1,000 0 25 50 75 100 125 150 WB: MCL150- Taxol (nM) Taxol (nM) WB: CDC27 CDC27-P CDC27 cdBax–/–Bak–/– Bcl2–/– Bax–/–Bak–/– 98- Bclw–/– Mcl1–/– WT WB: Tubulin 120 –/– –/– 50- WT Bclx 120 Bclx –/– Mcl1 FBW7–/– FBW7–/– FBW7–/– FBW7 100 100 d WT + FBW7α + FBW7β + vector + vector 80 80 Mitotic time course (h): –2 M +2+4+6 +8+10+12–2 M +2 +4 +6 +8 +10+12–2 M +2 +4 +6+8+10+12–2 M +2 +4 +6 +8 +10+12 60 60 50- WB: MCL1 40 40 Viability (%) Viability (%) WB: CDC27 CDC27-P 20 20 98- CDC27 WB: Tubulin 0 0 50- 0 200 400 600 800 1,000 0 200 400 600 800 1,000 e FBW7+/+ FBW7–/– FBW7+/+ FBW7–/– Taxol (nM) Vincristine (nM) Mitotic time course (h): –3 M +3 +6 +9 –3 M +3 +6 +9 –3 M +3 +6 +9 –3 M +3 +6 +9 Nocodazole Taxol e IP: MCL1 WB: CUL1 Antitubulin –1 WB: CUL1 98- 98- agent: (200 ng ml ) (200 nM) IP: MCL1 WB: SKP1 Mitotic time WB: SKP1 22- 22- course (h): –4 –2 M +3+6 +9 +12+15 –4 –2 M +3+6 +9 +12+15 IP: MCL1 WB: ROC1 WB: MCL1 WB: ROC1 6- 6- IP: MCL1 50- WB: CDC27 CDC27-P WB: MCL1 98- CDC27 WB: BCL2 WB: Tubulin 50- f WB: BCL-XL FBox FBW7 FBW7-Δ β-TRCPFBW7 HA–CUL1 WT DN Myc–FBox +++++–+ + WB: BAD Flag–MCL1 ++– +++ + ++++ + HA–CUL1 WT WT WT WT WT WT WT WT WT WT WT WT WT DN LysateElutionLysateElution E1 –++++++++ +++++ HA–WT UBCH5A +–++++++ ++++++ IP: HA WB: BAK - CUL1 WB: HA HA–DN CUL1 IP: HA WB: BAX IP: Flag WB: ROC1 ROC1 WB: Biotin MCL1–Ub WB: BIM - Myc–FBW7 IP: Myc Myc–FBW7- WB: NOXA WB: Myc ΔFBox β IP: Flag Myc– -TRCP WB: PUMA WB: Flag Flag–MCL1 IP: HA HA–WT CUL1 WB: HA WB: CDC27 CDC27-P HA–DN CUL1 CDC27 WB: Tubulin Figure 2 | SCFFBW7 targets MCL1 for proteasomal degradation during mitotic arrest.
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