Oncogene (2008) 27, 2999–3009 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE A novel function for HSF1-induced mitotic exit failure and genomic instability through direct interaction between HSF1 and Cdc20 YJ Lee1, HJ Lee1, JS Lee2, D Jeoung3, CM Kang1, S Bae1, SJ Lee2, SH Kwon4, D Kang4 and YS Lee1 1Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; 2Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea; 3College of Natural Sciences, Kangwon National University, Chunchon, Korea and 4Korea Basic Science Institute, Chunchon Center, Chunchon, Korea Although heat-shock factor (HSF) 1 is a known highly aneuploid, however, the molecular mechanisms transcriptional factor of heat-shock proteins, other path- underlying the development of aneuploidy have not wayslike production of aneuploidy and increasedprotein been fully defined, although mutations in mitotic stability of cyclin B1 have been proposed. In the present checkpoint genes have been identified in a subset of study, the regulatory domain of HSF1 (amino-acid human cancers and cell lines (Lengauer et al., 1997; sequence 212–380) was found to interact directly with Cahill et al., 1998). the amino-acid sequence 106–171 of Cdc20. The associa- Enhanced heat-shock protein (HSP) expression in tion between HSF1 and Cdc20 inhibited the interaction response to various stimuli is regulated by heat-shock between Cdc27 and Cdc20, the phosphorylation of Cdc27 factors (HSFs), the functional relevance of which is now and the ubiquitination activity of anaphase-promoting emerging. HSF1 (an HSF prototype) and HSF3 are complex (APC). The overexpression of HSF1 inhibited responsible for heat-induced HSP expression, whereas mitotic exit and the degradationsof cyclin B1 and securin, HSF2 is refractory to classical stressors, and HSF4 is which resulted in production of aneuploidy and multi- expressed in a tissue-specific manner (Pirkkala et al., nucleated cells, but regulatory domain-deficient HSF1 2001). Homology between the amino-acid sequences of did not. Moreover, HSF1-overexpressing cells showed human HSFs is only 30–40%, and evidence for specific elevated levelsof micronuclei and genomic alteration. The functions of HSFs beyond heat-shock response is depletion of HSF1 from cellshighly expressingHSF1 speculative. Many types of tumors contain high reduced nocodazole-mediated aneuploidy in cells. These concentrations of HSPs of the HSP27, HSP70 and findingssuggesta novel function of HSF1 frequently HSP90 families (Hoang et al., 2000; Cen et al., 2004). overexpressed in cancer cells, to inhibit APC/C activity The roles of HSPs in tumor development may also be by interacting with Cdc20, and to result in aneuploidy related to their functions in the development of development and genomic instability. tolerance to applied stress (Li and Werb, 1982). Oncogene (2008) 27, 2999–3009; doi:10.1038/sj.onc.1210966; However, the precise relationships between HSFs and published online 3 December 2007 HSPs during cancer development have not been clearly defined and relatively few papers about the role of HSF1 Keywords: HSF1; interaction with Cdc20; mitotic in cancer or carcinogenesis are available (Cen et al., arrest; aneuploidy; inhibition of APC activity 2004; Wang et al., 2004; Min et al., 2007). In this study, we identified a novel function of HSF1 with respect to inhibition of mitotic exit results in genomic instability and the development of aneuploidy and chromosomal missegregation, which it achieves by Introduction interacting directly with Cdc20 and thus inhibiting anaphase-promoting complex (APC) activity. More- Aneuploidy, which is defined as an abnormal number of over, these phenomena were found to be independent chromosomes or chromosome segments, is a ubiquitous of HSP induction, for example, of HSP25 and HSP70. feature of solid human tumors, and causes genetic instability and promotes further aneuploidy (Hida and Klagsbrun, 2005). The majority of cancer cells are Results Correspondence:Dr YS Lee, Division of Radiation Effect, Korea HSF1 binds directly to Cdc20 Institute of Radiological and Medical Sciences, 215-4 Gongneung- Because HSF1-transfected radiation-induced fibrosar- Dong, Nowon-Ku, Seoul 139-706, Republic of Korea. coma (RIF) cells showed large numbers of aneuploid E-mail:[email protected] Received 26 July 2007; revised 31 October 2007; accepted 31 October cells and mitotic arrest after radiation or heat-shock 2007; published online 3 December 2007 (data not shown), we examined whether HSF1 affect cell HSF1-induced mitotic exit failure and genomic instability YJ Lee et al 3000 cycle proteins that have the potential to regulate mitosis. of HSF1 (the N terminus of HSF1, HSF1DN) and of Immunoprecipitation (IP) analysis of HSF1 with several 380–503 of HSF1 (the C-terminal region, HSF1DC) mitotic regulators such as Cdc20, Cdc27 and Cdc26 and interacted with Cdc20, but deletion mutant of amino so on revealed that HSF1 interacted with Cdc20 in acids 212–380 of the HSF1 regulatory domain HSF1-overexpressing cells, while HSP25 did not (HSF1DReg) did not, as revealed by co-IP and (Figure 1a). An in vitro translation assay also indicated immunoblotting (Figure 1c). Moreover, the F2 deletion direct binding between HSF1 and Cdc20 (Figure 1b). mutant of Cdc20 (Cdc20DF2) did not interact with When the interaction between HSF1 and Cdh1, another HSF1, whereas the amino acids 1–171 (F1) and 108–171 APC partner in mitotic progression was examined, no (F2) bound directly to HSF1 (Figure 1d). These data interaction was observed, suggesting a specific interaction indicate that the interaction sites of HSF1 and Cdc20 between HSF1 and Cdc20 (Supplementary Figure 1A). are the regulatory domain of HSF1 and the F2 domain To elucidate whether other HSFs like HSF2 and 4 can of Cdc20. also bind to Cdc20, IP was performed after transfection of HSF1, 2 or 4. It was found that only HSF1 bound Cdc20. Moreover, when we transfected Cdc20 to human The interaction between HSF1 and Cdc20 inhibits APC- osteogenic sarcoma (HOS) cells that show high levels of Cdc20 binding and APC activity HSF1, 2 and 4, same result was obtained (Supplemen- The affinity of APC for activators is regulated by the tary Figure 1B). Deletion mutants of amino acids 1–212 phosphorylations of APC subunits (that is, APC1, RIF-HA-pcDNA3RIF-HA-HSF1RIF-HSP25 HA-pcDNA3HA-HSF1HA-HSP25 75 GST-Cdc20 protein + + + HA 75 25 HSP25 37 GST pull down 35S 50 75 IP: Cdc20 37 25 50 IB: HSF1 75 75 IP: Cdc20 HSF1 35 37 In vitro translated IB: HA S 35 50 50 S-labeled protein IP: Cdc20 HSP25 37 25 25 IB: HSP25 N-terminal C-terminal N-terminal C-terminal 1 106 171 499 1 212 380 503 Cdc20 HSF1 F1 HSF1∆C F2 HSF1∆N ∆F2 HSF1∆Reg HA-HSF1 - + + + + + 499 GST - + - - - - GST-Cdc20 - - + - - - N C ∆ ∆ ∆Reg GST-Cdc20∆F2 - - - + - - SF1 SF1 SF1 SP25 GST-Cdc20F1 - - - - + - pcDNA3HA-HSF1HA-H HA-H HA-H HA-H GST-Cdc20F2 - - - - - + IP:Cdc20 IB:HA IP: GST IB: HA HSF1 HA GST-Cdc20 GST-Cdc20∆F2 75 GST GST-Cdc20 F1 50 CDC20 GST-Cdc20F2 37 GST GST Figure 1 Heat-shock factor 1 (HSF1) interacts with Cdc20. (a) Lysates of radiation-induced fibrosarcoma (RIF) cells expressing hemagglutinin (HA)-HSF1 or HA-HSP25 were immunoprecipitated with anti-Cdc20, and the resulting immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (b) Glutathione S-transferase (GST) pull-down assays were performed using TNT kits by mixing GST-Cdc20 fusion proteins (upper) and in vitro translated 35S-labeled proteins (pcDNA3, HSF1 and HSP25; lower). (c) Anti-Cdc20 immunoprecipitates prepared from RIF cells transfected with HA-HSF1 mutants were analysed using anti-HA and anti-Cdc20. (d) Anti-GST immunoprecipitates prepared from RIF cells transfected with GST-Cdc20 mutants were analysed using anti-HA and anti-GST. Oncogene HSF1-induced mitotic exit failure and genomic instability YJ Lee et al 3001 Cdc27, Cdc16 and Cdc23) (Cross, 2003). To elucidate Cdc20 and Cdc27 during mitosis. IP analysis also whether HSF1-Cdc20 binding affects the Cdc20–APC revealed that Cdc20–Cdc27 binding was diminished interaction and the phosphorylations of APC subunits, after nocodazole treatment in HSF1-overexpressing HSF1 or HSP25 proteins were added to lysates of cells. However, control vector-transfected cells showed nocodazole-treated cells. As shown in Figure 2a (left), a higher level of Cdc20–Cdc27 interaction after release the interaction between Cdc20 and Cdc27, augmented from nocodazole block and increased phosphorylation by nocodazole treatment over a 16 h period, was of Cdc27 was reduced by HSF1 transfection (Figure 2a, reduced when HSF1 protein was added to the initial right). We next examined whether HSF1 affects the mix, but not when HSP25 protein was added, suggesting ubiquitination activity of APC by associating with that HSF1 directly inhibited the interaction between Cdc20. The ubiquitin ligase activity of APC in mitotic HA-pcDNA3 HA-HSF1 Nocodazole Con 0 30 90 150 Con 0 30 90 150 release (min) Nocodazole - + - + - + HSP25 protein - - + + - - IP: Cdc20 HSF1 protein - - - - + + IB: Cdc27 IP: Cdc20 IB: Cdc27 Cdc20 HSF1 HA HSP25 HA-pcDNA3 HA-HSF1 Nocodazole Cdc20 Cont 0 30 90 150 Cont 0 30 90 150 release (min) p-Cdc27 Cdc27 Cdc27 Cdc27 HA β-Actin ∆Reg SF1 HSP25 - 4ug HSF1- 2ug 4ug HA-pcDNA3HA-HSF1HA-H Cdc20 ++ Cdc20 +++ APC ++ APC +++ HA 150 150 100 Ub-conjugates 100 150 75 IP: CyclinB1 75 100 IB: Ub S35-Cyclin B1 75 50 50 Ub-Conjugates Input Cdc20 Cdc20 50 CyclinB1-ub IP: CyclinB1 Input HSF1 HSP25 IB: CyclinB1 Figure 2 Heat-shock factor 1 (HSF1) to Cdc20 binding inhibits anaphase-promoting complex/cyclosome (APC/C) to Cdc20 binding. (a) Cell extracts from RIF cells (1 mg) pretreated with nocodazole (100 nM) for 16 h were incubated with or without HSP25 protein (10 mg) or HSF1 protein (10 mg).