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RASSF1A Interacts with and Activates the Mitotic Kinase Aurora-A

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Oncogene (2008) 27, 6175–6186 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE RASSF1A interacts with and activates the mitotic kinase Aurora-A L Liu1, C Guo1, R Dammann2, S Tommasi1 and GP Pfeifer1 1Division of Biology, Beckman Research Institute, City of Hope Cancer Center, Duarte, CA, USA and 2Institute of Genetics, University of Giessen, Giessen, Germany The RAS association domain family 1A (RASSF1A) gene tumorigenesis and carcinogen-induced tumorigenesis is located at chromosome 3p21.3 within a specific area of (Tommasi et al., 2005; van der Weyden et al., 2005), common heterozygous and homozygous deletions. RASS- supporting the notion that RASSF1A is a bona fide F1A frequently undergoes promoter methylation-asso- tumor suppressor. However, it is not fully understood ciated inactivation in human cancers. Rassf1aÀ/À mice how RASSF1A is involved in tumor suppression. are prone to both spontaneous and carcinogen-induced The biochemical function of the RASSF1A protein is tumorigenesis, supporting the notion that RASSF1A is a largely unknown. The homology of RASSF1A with the tumor suppressor. However, it is not fully understood how mammalian Ras effector novel Ras effector (NORE)1 RASSF1A is involved in tumor suppression pathways. suggests that the RASSF1A gene product may function Here we show that overexpression of RASSF1A inhibits in signal transduction pathways involving Ras-like centrosome separation. RASSF1A interacts with Aurora-A, proteins. However, recent data indicate that RASSF1A a mitotic kinase. Surprisingly, knockdown of RASS- itself binds to RAS only weakly and that binding to F1A by siRNA led to reduced activation of Aurora-A, RAS may require heterodimerization of RASSF1A and whereas overexpression of RASSF1A resulted in in- NORE1 (Ortiz-Vega et al., 2002). In addition, there is creased activation of Aurora-A, suggesting that RASS- evidence for an association of both NORE1 and F1A is involved in Aurora-A activation. Like other RASSF1A with the pro-apoptotic kinase MST1 and Aurora-A activators, RASSF1A was also a substrate of that this interaction is involved in apoptosis induced by Aurora-A in vitro. The failure of recombinant RASSF1A activated Ras (Khokhlatchev et al., 2002). to activate recombinant Aurora-A indicates that RASS- Recently, several groups, including ours, have re- F1A may not activate Aurora-A directly and suggests that ported that RASSF1A is a microtubule- and centro- RASSF1A may function as a scaffold to bring together some-associated protein, which regulates mitotic Aurora-A and its activator(s). Inhibition of centrosome progression (Liu et al., 2003, 2005c; Dallol et al., 2004; separation by RASSF1A overexpression is most likely a Rong et al., 2004; Song et al., 2004; Vos et al., 2004). We consequence of hyperstabilization of microtubules by this have shown that overexpressed RASSF1A co-localized protein. with microtubules in interphase cells and with spindles Oncogene (2008) 27, 6175–6186; doi:10.1038/onc.2008.220; and centrosomes during mitosis (Liu et al., 2003). published online 21 July 2008 Forced expression of RASSF1A induced mitotic arrest with aberrant mitotic cells (Liu et al., 2003). Similar Keywords: RASSF1A; Aurora-A; centrosome; mitosis results been have been obtained by several other groups (Dallol et al., 2004; Rong et al., 2004; Song et al., 2004; Vos et al., 2004). Song et al. (2004) have reported that RASSF1A regulated mitosis by inhibiting the anaphase- Introduction promoting complex (APC) through binding to CDC20 and induces G2–M arrest at pro-metaphase. The The RAS association domain family 1A (RASSF1A) function of RASSF1A was independent of the protein gene is located at chromosome 3p21.3 within a specific early mitotic inhibitor 1, and therefore Song et al. (2004) area of common heterozygous and homozygous dele- proposed that RASSF1A acted in early prometaphase tions (Dammann et al., 2000; Lerman and Minna, 2000). to prevent the degradation of mitotic cyclins and to The RASSF1A gene is one of the most frequently delay mitotic progression. However, our laboratory has methylation-silenced genes described in human cancer been unable to confirm an interaction between RASS- to date (Agathanggelou et al., 2005; Dammann et al., F1A and CDC20 (Liu et al., 2007). RASSF1A may 2005). Rassf1aÀ/À mice are prone to both spontaneous function as a tumor suppressor through promoting apoptosis and/or by controlling mitotic cell division (Agathanggelou et al., 2005; Dammann et al., 2005). Correspondence:Dr GP Pfeifer, Division of Biology, Beckman However, its exact mechanism of action remains to be Research Institute, City of Hope Cancer Center, City of Hope, 1500 determined. East Duarte Road, Duarte, CA 91010, USA. E-mail:[email protected] Aurora-A belongs to a family of highly conserved Received 14 April 2008; revised 4 June 2008; accepted 10 June 2008; serine/threonine kinases, which includes Aurora-A, -B published online 21 July 2008 and -C in mammals. Aurora kinases combined with RASSF1A interacts with and activates Aurora-A LLiuet al 6176 cyclin-dependent kinase 1 (or p34cdc2), Polo-like kinases 2005; Weaver and Cleveland, 2006). Being a putative and NIMA-related kinases are four families of known oncogene, Aurora-A has attracted a great deal of mitotic kinases that regulate mitotic progression. All attention as a potential antitumor target (Keen and three members of the Aurora kinase family share a Taylor, 2004; Mortlock et al., 2005; Naruganahalli conserved C-terminal catalytic domain and have been et al., 2006). found to have multiple mitotic functions (Andrews Here we demonstrate that endogenous RASSF1A et al., 2003; Carmena and Earnshaw, 2003). Among interacts with and activates Aurora-A. them, Aurora-A is the most intensively studied one. Originally Aurora-A was identified in a screen for Drosophila melanogaster mutants that have abnormal spindle-pole phenotypes (Glover et al., 1995). Aurora-A Results is located on centrosomes from late S/early G to the 2 RASSF1A regulates centrosome separation time when the cells exit mitosis (Carmena and Earn- shaw, 2003). Aurora-A is important in centrosome RASSF1A is a microtubule- and centrosome-associated maturation and separation as well as in spindle assembly protein in interphase cells and is found at the spindle et al (Andrews et al., 2003; Carmena and Earnshaw, 2003; poles and midzone region in mitotic cells (Liu ., et al et al Marumoto et al., 2005). Moreover, activity of Aurora-A 2003, 2005a, c; Dallol ., 2004; Rong ., 2004; Vos et al et al is required for timely mitotic entry (Marumoto et al., ., 2004; van der Weyden ., 2005). Endogenous 2002, 2003; Hirota et al., 2003; Hachet et al., 2007; RASSF1A is primarily a centrosomal protein in et al Portier et al., 2007). Both upregulation and down- interphase cells (Guo ., 2007) and it colocalizes regulation of Aurora-A results in cytokinesis failure with mitotic spindles in mitosis (Figure 1a). Using (Meraldi et al., 2002; Anand et al., 2003; Marumoto EGFP-tagged deletion fragments of RASSF1A, we et al., 2003; Zhang et al., 2004), indicating that the determined that the centrosome and spindle pole timing of Aurora-A activity is important for proper localization of RASSF1A requires a minimal domain cytokinesis. fragment from amino acid D120 to G193 (Figure 1b). Expression and localization of Aurora-A is cell-cycle This domain is distinct from the RAS association regulated. Its protein level peaks in the middle of mitosis domain present in RASSF1A (R194–S288) and only and is diminished in late mitosis/early G by the Cdh1- partially overlaps with the minimum microtubule- 1 et al activated APC/C (APC/cyclosome) (Honda et al., 2000; binding domain (D120-S288) (Liu ., 2003). Castro et al., 2002; Taguchi et al., 2002). The activity of Previously, we found that overexpression of RASS- Aurora-A is also controlled by phosphorylation. Phos- F1A resulted in over 50% of mitoses with monopolar et al phorylation of Thr288 of human Aurora-A is essential spindles (Liu ., 2003). Generally, monopolar for its kinase activity (Walter et al., 2000; Littlepage spindles may result from defects in centrosome duplica- et al., 2002). Several activators of Aurora-A have been tion or from failure of centrosome separation. When identified, which include TPX2, Ajuba, HEF1, Bora and these cells were stained with an anti-g-tubulin antibody, PAK1 (Kufer et al., 2002; Hirota et al., 2003; Crane we observed that there were two closely spaced g-tubulin et al., 2004; Satinover et al., 2004; Pugacheva and spots in 96% of the monopolar spindle containing cells, Golemis, 2005; Zhao et al., 2005; Hutterer et al., 2006). which indicates that the centrosomes have duplicated Activation of Aurora-A by TPX2 is required for but were not separated far enough to organize a bipolar bipolar-spindle assembly (Trieselmann et al., 2003; Tsai spindle (Figure 2). et al., 2003), whereas activation of Aurora-A by Ajuba is Microtubules are important in centrosome separation et al et al et al essential for mitotic entry (Hirota et al., 2003). It is (Waters ., 1993; Gaglio ., 1996; Uzbekov ., tempting to propose that Aurora-A activators may at 2002). Therefore, we speculated that inhibition of least partially control the temporal and spatial distribu- microtubule dynamics by RASSF1A may at least in tion of Aurora-A activity along the cell division cycle. part account for its effect on centrosome separation. Aurora-A is frequently overexpressed in various Indeed, the monopolar spindles were hyperstabilized human tumors (Bischoff et al., 1998; Zhou et al., and resistant to nocodazole-induced depolymerization 1998). Overexpression of Aurora-A was sufficient to (Figure 2). transform NIH3T3 and Rat1 cells (Bischoff et al., 1998; Zhou et al., 1998). However, the same regimen did not RASSF1A interacts with Aurora-A transform primary murine embryonic fibroblasts We asked if there are other mechanisms by which (MEFs), suggesting that upregulation of Aurora-A itself RASSF1A may regulate centrosome separation.
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  • Discovery of BPR1K871, a Quinazoline Based

    Discovery of BPR1K871, a Quinazoline Based

    www.impactjournals.com/oncotarget/ Oncotarget, 2016, Vol. 7, (No. 52), pp: 86239-86256 Research Paper Discovery of BPR1K871, a quinazoline based, multi-kinase inhibitor for the treatment of AML and solid tumors: Rational design, synthesis, in vitro and in vivo evaluation Yung Chang Hsu1,*, Mohane Selvaraj Coumar2,*, Wen-Chieh Wang1,*, Hui-Yi Shiao1,*, Yi-Yu Ke1, Wen-Hsing Lin1, Ching-Chuan Kuo1, Chun-Wei Chang1, Fu-Ming Kuo1, Pei-Yi Chen1, Sing-Yi Wang1, An-Siou Li1, Chun-Hwa Chen1, Po-Chu Kuo1, Ching- Ping Chen1, Ming-Hsine Wu1, Chen-Lung Huang1, Kuei-Jung Yen1, Yun-I Chang1, John T.-A. Hsu1, Chiung-Tong Chen1, Teng-Kuang Yeh1, Jen-Shin Song1, Chuan Shih1, Hsing-Pang Hsieh1,3 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan, ROC 2Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, India 3Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, ROC *These authors contributed equally to this work Correspondence to: Hsing-Pang Hsieh, email: [email protected] Keywords: acute myeloid leukemia, aurora kinase, FLT3, quinazoline, multi-kinase inhibitor Received: July 25, 2016 Accepted: November 07, 2016 Published: November 15, 2016 ABSTRACT The design and synthesis of a quinazoline-based, multi-kinase inhibitor for the treatment of acute myeloid leukemia (AML) and other malignancies is reported. Based on the previously reported furanopyrimidine 3, quinazoline core containing lead 4 was synthesized and found to impart dual FLT3/AURKA inhibition (IC50 = 127/5 nM), as well as improved physicochemical properties. A detailed structure-activity relationship study of the lead 4 allowed FLT3 and AURKA inhibition to be finely tuned, resulting in AURKA selective (5 and 7; 100-fold selective over FLT3), FLT3 selective (13; 30-fold selective over AURKA) and dual FLT3/AURKA selective (BPR1K871; IC50 = 19/22 nM) agents.