Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells

Abstract

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.

Figure 1

NSC305787 and NSC668394 directly interact with ezrin. (a) WT and T567D ezrin proteins are expressed in bacteria. The most prominent band in bacterial lysates following isopropyl-beta-D-thiogalactopyranoside induction appeared just above the 75-kD molecular weight marker on a 10% polyacrylamide gel stained with coomassie blue (lanes 2 vs 3 and 5 vs 6). The best fractions from column chromatography purifications for each protein are shown in lanes 4 and 7 (arrowhead). (b) T567D ezrin shows stronger binding to actin than WT. K12 cell lysate was incubated with WT and T567D ezrin proteins, followed by immunoprecipitation with ezrin antibody and immunoblotting with ezrin and actin antibodies. Control (ctrl) lane is K12 lysate without recombinant protein treatment. (c) Chemical structures of NSC305787 and NSC668394 are given. (d) Direct binding of NSC305787 to WT ezrin protein was analyzed by SPR. Compound was injected at 1-, 2-, 4-, 8-, 32- and 64-μM concentrations in duplicates. A representative set of binding curves are presented with the steady-state affinity curve given in the inset. Red Xs represent binding levels that were used to calculate the steady state affinity in the inset graph. (e) NSC668394 was injected at 1.5-, 3-, 6-, 12.5-, 25-, 50- and 100-μM concentrations in triplicates. Black lines show actual data points, and red lines show curve fits for 1:1 binding model. Mean K_D values and standard deviations were calculated from five independent experiments for each compound.

Figure 2

NSC305787 and NSC668394 inhibit ezrin T567 phosphorylation. (a, b) Effect of compounds on recombinant ezrin phosphorylation by recombinant PKCΙ was tested in an in vitro kinase assay. Experiments were repeated three times, and densitometric analysis of bands was used for calculation of relative kinase activity (graphs). Error bars represent the s.d. from three independent experiments. Kinase activities of PKCΙ, α and γ were also evaluated with a non-specific substrate (myelin basic protein) in the presence of NSC305787 and NSC668394 using a radioactive in vitro kinase assay. (c) Both compounds inhibit phosphorylation of endogenous ezrin protein and interaction with actin without altering cellular ezrin levels. K7M2 cells were treated with NSC305787 (10 μM) and NSC668394 (10 μM) for 6 h and then subjected to immunoprecipitation with ezrin antibody followed by immunoblotting with phosphoezrin, actin and ezrin antibodies. Control (Ctrl) lane is DMSO (1%)-treated K7M2 lysate.

Figure 3

NSC305787 and NSC668394 inhibit ezrin-mediated invasion of K7M2 OS cells. (a) Immunoblotting showed that the K7M2 cell line had higher levels of ezrin protein expression than K12 cells. (b, c) The anti-invasive potential of NSC305787 and NSC668394 was validated in K7M2 and K12 cells using an electric cell impedance system. DMSO (1%) was used as a control. Error bars represent standard deviations from duplicate data points. In this assay, a decrease in cell index represents invasion of the HUVEC monolayer by OS cells.

Figure 4

NSC305787 and NSC668394 cause reduced cell motility phenotypes in zebrafish. (a) In the late blastula period, blastodisc cells begin to spread over the yolk cell in the process of epiboly. Reduction of ezrin protein levels by anti-sense MO results in epiboly defects characterized by cells not spreading over the yolk cell but instead piling up at the animal pole. MO-injected embryos were scored for epiboly defects at the 90% epiboly stage: WT (un-injected), 4/73 (5%); 3 ng MO, 5/12 (42%); 6 ng MO, 33/48 (69%); and 12 ng MO, 20/29 (69%). (b) When embryos were treated with 10-μM NSC305787, 12 of 81 embryos (15%) exhibited epiboly defects that completely mimicked MO-injected embryos. At 20-μM NSC305787, 71 of 71 embryos (100%) demonstrated epiboly defects. (c) Normal eye development follows lateral movement of progenitor cells to form two eyes in WT embryos. NSC668394 (10 μM) inhibited motility of eye progenitor cells, resulting in cycloptic embryos at 28 hpf in 97 of 99 embryos (98%). (d) NSC668394-treated embryos continued to grow and formed a single functional eye at 6 days after fertilization. (e) Percentages of animals with the observed phenotypes upon treatment are indicated.

Figure 5

NSC305787 and NSC668394 inhibit OS metastatic growth in lung organ culture. (a) GFP-expressing K7M2 OS cells were injected into tail veins of BALB/C mice, and the lungs were dissected. Lung slices were grown in culture media, and tumor growth was monitored. Quantitation of the fluorescence signal from NSC305787 (10 μM), NSC668394 (10 μM) and vehicle (DMSO, 1%)-treated organ cultures. Data are represented as means±s.d. from three independent experiments. (* P<0.05, ** P<0.005, using a two-tailed Student’s t-test) (b) Fluorescence images of a representative culture treated with NSC305787, NSC668394 and vehicle are shown.

Figure 6

NSC305787 and NSC668394 inhibit in vivo OS metastatic growth in lungs. (a) Kaplan–Meier survival curves show the percentage survival for NSC305787 and NSC668394 treatment over time in K7M2-injected animals. The overall survival of NSC305787-treated mice was significantly different than that of the vehicle-treated group and NSC668394-treated mice showed an increase in survival, but this increase was not statistically significant from the vehicle group (P-value of NSC305787 is 0.0337 and NSC668394 is 0.0524). Log-rank statistics were used for survival curves. (b) Representative fluorescence images of whole lungs at the time of necropsy are shown. (c) Survival ratios of the groups were not different because MNNG-HOS is an ezrin-independent cell line. (d) Representative fluorescence images of whole lungs from MNNG-HOS-injected animals at the time of necropsy are shown.