Small molecule targeting the Hec1/Nek2 mitotic pathway suppresses tumor cell growth in culture and in animal

Abstract

Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI(50), 10-21 micromol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds.

Figure 1

Identification of small compounds that disrupt Hec1/Nek2 interaction A, Schematic diagram of a reverse yeast-two hybrid system for high throughput screening. TetR fused with the C-terminal binding region of Hec1 (TetR-Hec1) was constitutively expressed; activation domain of GAL1 fused with Nek2 (AD-Nek2) was under the control of GAL1 inducible promoter. If an inhibitor abolishes the Hec1/Nek2 interaction, 5-FOA (5-fluoroorotic acid) will not be metabolized, thus permitting yeast growth; otherwise, 5-FOA will be hydrolyzed into toxic metabolites and inhibits yeast growth. B, structures of two candidate compounds that promote yeast growth.

Figure 2

INH1 compound binds to Hec1, but not Nek2. A–B, the Surface Plasma Resonance (SPR) assays. A, Schematics showing that either Hec1 or Nek2 can be first immobilized on the chip. Protein-bound chip is pretreated with candidate compound or mock before the second protein is added. B, Histograms to show retained resonance units (RU) with Hec1-immobilized chip (left) or Nek2-immobilized chip (right). Full length Hec1 and Nek2, tagged with 6xHis and GST respectively, were purified from bacteria. C, (left) structures of INH1-conjutated and control matrix. Grey balls denote the Affigel-10 matrix; (right) Affinity-pulldown by using INH1 or control -conjugated matrix to incubate with HeLa cell extract. Matrix bound proteins were further competed with free INH1 of varying doses in the reaction (lanes 3–5). Resultant precipitates were resolved by SDS-PAGE and detected for Hec1 or Nek2 by Western blotting. D, co-immunoprecipitation/Western blot assays with cells treated with DMSO (mock) or INH1 (25 μM, 36 hrs).

Figure 3

INH1 treatment triggered Nek2 reduction and defective Hec1 localization on kinetochores. A, Western blotting analysis of cellular Hec1, Nek2 and cyclin B after INH1 treatment. B, Western blotting for cellular Hec1, Nek2 and Rad50 (loading control) after Hec1 depletion by RNAi transfection. Luc, control luciferase RNAi; Hec #1 and #2 refer to two distinct RNAi sequences (31, 34). C, quantitative immunofluorescent staining of INH1 or mock treated mitotic cells with anti-Hec1 or anti-ACA antibodies. The relative immunofluorescence intensity (Hec1 vs. ACA) was shown in the bar graph (D). Scale bar, 10 μm.

Figure 4

INH1 effectively inhibits the proliferation of human breast cancer lines. A, Table shows GI₅₀s (50% growth inhibition) of INH1 on a panel of human breast cancer cell lines, SV40-immortalized breast epithelial cell line HBL-100, and noncancerous breast epithelial cell line MCF10A. B, Colonogenic assays for MCF7 and MDA-MB-468 cells treated with various doses of INH1. Bar graph shows the colony number.

Figure 5

Cellular phenotypic changes of INH1-treated HeLa cells. A, Panels a to e are representative phase-contrast images of HeLa cells treated with INH1. Nucleus/DNA of control cells (f) or INH1-treated cells (g) (10 μM, 72 hrs) were stained with DAPI and arrows in (g) indicate condensed nuclear globules, characteristic of apoptosis. Scale bars, 20 μm (black) or 10 μm (white). B, cell cycle profiling of INH1-treated cells by FACS analysis. Sub-G1 populations were denoted by arrows. C, time-dependent kinetics showing the percentages of mitotic HeLa-H2B-GFP cells with metaphase misalignment after INH1 treatment (10 μM). Representative images of mitotic chromosomes stained with DAPI after treatment with either DMSO (a–c) or INH1 (10 μM) for 32 hrs (d–f). Arrows show misaligned (d–e) or lagging (f) chromosomes. Scale bar, 10 μm. D, immunofluorescent staining with anti-α–tubulin and γ-tubulin to show the normal (row a; arrows) and abnormal (rows b &c; arrows) spindle morphology. Scale bar, 10 μm.

Figure 6

INH1 inhibits breast tumor growth in a xenografted mouse model. A, Changes of the tumor volumes following the INH1 treatment. MDA-MB-468 cells were injected into athymic nude mice under the mammary fat-pad 10 days before drug treatment; then these tumor bearing nude mice (N=7 mice for each group) were treated with control vehicle, 50 mg/kg or 100 mg/kg (body weight) of INH1 every other day for 7 weeks (I.P. injection, 25 cycles). Tumor volumes were measured over time and plotted in the graph. The p values were derived from the ANOVA test. B, the histogram showing the body weight of the mice treated in (A).