Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules

Abstract

The overexpression of Aurora kinases in multiple tumors makes these kinases appealing targets for the development of anticancer therapies. This study identified two small molecules with a furanopyrimidine core, IBPR001 and IBPR002, that target Aurora kinases and induce a DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study used the IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation, thereby revealing the coexistence and continuous cycling of HURP between unphosphorylated and phosphorylated forms that are associated, respectively, with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference for HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool for the discovery of new cell-division molecules for the development of antitumor drugs.

Fig. 1.

Structures of IBPR compounds that induce a change in DFG conformation in Aurora A. (A) Chemical structures of IBPR001 (1) and IBPR002 (2). (B) IC₅₀ of the compounds from the in vitro Aurora A and Aurora B activity assay. (C) Active sites of Aurora A in complex with IBPR001 and VX-680. The interacting residues with the inhibitors are shown in stick representation and are labeled. The DFG motif (cyan) at the activation loop (A-loop, yellow) adopts a different conformation to accommodate IBPR001 from VX-680. Hydrogen bonds between the inhibitors and Aurora A are shown as red dashed lines. For figure presentation clarity, hydrogen bonds between the inhibitors and the hinge region are omitted. (D) Athymic nude mice xenograft with HCT116 cancer cells were injected i.v. with control vehicle or 50 mg/kg of VX-680 or IBPR002. Mean tumor volumes (in cubic millimeters) ± SEM (n = 10 per group) are shown from the initiation of treatment (∼100 mm³). *P < 0.05 compared with vehicle.

Fig. 2.

IBPR001 and IBPR002 efficiently eliminate HURP phosphorylation. (A) Immunoblot of HURP from HeLa cells treated with increasing concentrations of the Aurora inhibitors VX-680, IBPR001, IBPR002, MLN8237, or AZD1152. Cells were arrested in the M phase with nocodazole (400 nM) for 24 h followed by 1-h cotreatment with the compounds and MG132 (5.0 μg/mL). Actin immunoblot was included as a loading control. Cyclin B1 (expressed in late G2-metaphase), pituitary tumor transforming gene (PTTG) (expressed in prophase-metaphase), and cyclin E1 (expressed in G1-S phase) (Fig. S2A) immunoblots are shown for verification of the M-phase synchronization. The Western signals of HURP-P and HURP-U are denoted. (B) Ratios of HURP-P/total HURP were quantified from the Western blot results in A. (C) The kinase activity of Aurora A for HURP phosphorylation was assessed in an in vitro kinase assay. Full-length FLAG-tagged HURP was expressed and immunoprecipitated from 293T cells as a substrate for Aurora A. The addition of active Aurora A increased the molecular size of HURP (compare lanes 1 and 5). The molecular size of HURP failed to increase in the reactions that contain MLN8237 (lane 3) and IBPR001 (lane 4) but increased in the reaction that contained AZD1152 (lane 2).

Fig. 3.

IBPR compounds and MLN8237 disrupt nucleation of kinetochore microtubules. (A) HeLa cells were treated as shown in the upper scheme, followed by coimmunofluorescence staining with two combinations of antibodies: HURP/α-tubulin/CREST (a centromere marker) or HURP/α-tubulin/Pericentrin (a centrosome marker). Tubulins were nucleated as thick bundles that link to kinetochores (i.e., K-fibers) in control, but not in IBPR001 or MLN8237 treated cells. DNA was stained with Hoechst33342. Noc, nocodazole. All images are summations of z-stacks. (Scale bars: 5 μm.) (B) Immunoblot of HURP from nocodazole-arrested HeLa cells treated with 1.0 μM of IBPR001 or MLN8237 and 5 μg/mL MG132. Total cell lysates were collected at 0, 30, 60, 90, and 120 min after the compound treatment. (C) Statistics of HURP that form K-fibers in A. (Scale bars: 5 μm.)

Fig. 4.

IBPR compounds restrict the association of HURP with centrosomal microtubules. (A) Representative HURP morphological phenotypes in HeLa cells treated with DMSO control (Left) or 1.0 μM of IBPR001/IBPR002 (Right) for 13 h following thymidine release. Cells were coimmunostained with rabbit anti-HURP and mouse anti–α-tubulin antibodies. DNA was stained with Hoechst33342. (B) Statistics of the representative HURP morphological phenotypes presented in A. (C) Cells were treated as shown in the upper scheme. HURP is associated with centrosomal microtubules (stained with α-tubulin) emanating from centrosomes (stained with Pericentrin) in IBPR001-treated cells upon nocodazole removal. Images are summations of z-stacks. (Scale bars: 5 μm.) (D) Cells were treated as in the upper scheme in C. Cell lysates were harvested every 30 min after the removal of nocodazole. Immunoblots of cyclin B1 and actin were included to indicate the cell-cycle status and serve as a loading control, respectively. The immunoblot of HURP shows that HURP remained unphosphorylated upon nocodazole removal (lanes 7–9). On the other hand, part of HURP was converted to the unphosphorylated form upon nocodazole removal in control cells (lanes 2–4, denoted by asterisks). (E) Immunoblot of HURP in nocodazole-arrested cells treated with 1.0 μM of IBPR001, 100 nM of Calyculin A, or both for 1 h. Immunoblot of actin was included as a loading control. (F) Cellular localization of HURP (green) and α-tubulin (red) in nocodazole-arrested cells treated with 1.0 μM of IBPR001 or cotreated with 1.0 μM of IBPR001 and 100 nM of Calyculin A. Cells were fixed after 1 h of drug treatment. A control cell (DMSO) is shown for comparison. Images are summation of z-stacks. (Scale bars: 5 μm.)

Fig. 5.

HURP-U is preferentially associated with the mother centrosome. (A) The eldest mother centriole was stained positive for Cenexin1. Centrosomes were stained positive for γ-tubulin. DNA was stained with Hoechst33342. (B) HURP preferentially resides with the mother centrosome that was stained positive (or stronger) for Cenexin1 in cells treated with 1.0 μM IBPR001 or MLN8237. Note that the γ-tubulin antibody stained both centrosomes. (C) Cells were treated with DMSO control or IBPR001 as shown in the scheme in B and were coimmunofluorescence stained for EG5, HURP, and TPX2. Images are maximum projections of z-stacks. (Scale bars: 5 μm.)

Fig. 6.

Models for nucleation of centrosomal and kinetochore microtubule fibers by Aurora A-regulated HURP phosphorylation during spindle formation. (A) As a cell enters mitosis, the nuclear envelope (light blue circle) breaks down; HURP is expressed and associated with the minus end of centrosomal microtubules that project toward chromosomes. As the cell cycle proceeds to prometaphase and metaphase, HURP gradually is phosphorylated and translocated to the vicinity of chromosomes to assist nucleation and stabilization of kinetochore fibers. Finally, bipolarity is established. Phosphorylated HURP forms a rod-like structure (purple bar) that links to kinetochore. (B) Treatment with 300–400 nM nocodazole enriched the phosphorylated HURP that nucleates kinetochore microtubules. (C) Adding IBPR001/IBPR002/MLN8237 disrupts nucleation of the kinetochore microtubule. (D) Removing nocodazole under the treatment with IBPR001/IBPR002/MLN8237 reinitiates tubulin polymerization from centrosomes but not from kinetochores. HURP goes to the minus end of centrosomal microtubules that face toward the chromosomes. (E) Inhibiting HURP phosphorylation by IBPR001/IBPR002/MLN8237 abolishes nucleation of HURP in the vicinity of chromosomes. The unphosphorylated HURP distributes restrictively to the minus end of centrosomal microtubules. Because IBPR001/IBPR002/MLN8237 do not completely block the separation of the duplicated centrosomes, HURP is preferentially associated with the microtubules that emanate from the mother centrosome, which resides in proximity to the chromosomes. (F) Models for HURP phosphorylation and dephosphorylation with and without nocodazole. In the presence of nocodazole, the force that drives HURP phosphorylation (by Aurora A) overrides dephosphorylation (through a PP1/PP2A-dependent pathway). Conversely, the ratio of phosphorylation/dephosphorylation is decreased in the absence of nocodazole.