Characterization of the biological activity of a potent small molecule Hec1 inhibitor TAI-1

Abstract

Background: Hec1 (NDC80) is an integral part of the kinetochore and is overexpressed in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. A highly potent first-in-class compound targeting Hec1, TAI-1, was identified and is characterized in this study to determine its potential as an anticancer agent for clinical utility.

Methods: The in vitro potency, cancer cell specificity, synergy activity, and markers for response of TAI-1 were evaluated with cell lines. Mechanism of action was confirmed with western blotting and immunofluorescent staining. The in vivo potency of TAI-1 was evaluated in three xenograft models in mice. Preliminary toxicity was evaluated in mice. Specificity to the target was tested with a kinase panel. Cardiac safety was evaluated with hERG assay. Clinical correlation was performed with human gene database.

Results: TAI-1 showed strong potency across a broad spectrum of tumor cells. TAI-1 disrupted Hec1-Nek2 protein interaction, led to Nek2 degradation, induced significant chromosomal misalignment in metaphase, and induced apoptotic cell death. TAI-1 was effective orally in in vivo animal models of triple negative breast cancer, colon cancer and liver cancer. Preliminary toxicity shows no effect on the body weights, organ weights, and blood indices at efficacious doses. TAI-1 shows high specificity to cancer cells and to target and had no effect on the cardiac channel hERG. TAI-1 is synergistic with doxorubicin, topotecan and paclitaxel in leukemia, breast and liver cancer cells. Sensitivity to TAI-1 was associated with the status of RB and P53 gene. Knockdown of RB and P53 in cancer cells increased sensitivity to TAI-1. Hec1-overexpressing molecular subtypes of human lung cancer were identified.

Conclusions: The excellent potency, safety and synergistic profiles of this potent first-in-class Hec1-targeted small molecule TAI-1 show its potential for clinically utility in anti-cancer treatment regimens.

Figure 1

Structure of Hec1 Inhibitor TAI-1.

Figure 2

TAI-1 Disrupts Hec1-Nek2 interactions, induces chromosomal misalignment and induces apoptosis of cancer cells. (A) K562 cells were treated with 500 nM TAI-1, lysates immunoprecipitated with anti-Nek2 antibody were probed for Hec1 by western blotting to determine interaction. (B) K562 cells were treated with TAI-1 at 1 μM for the indicated time points and collected for immunoblotting of Hec1 and Nek2. (C) MDA-MB-468 cells treated with 1 μM TAI-1 were immunofluorescent stained for DNA and mitotic spindle. (D) Metaphase cells were counted for percentage of cells with misaligned chromosomes. (E) Lysates of HeLa treated with TAI-1 for 8 or 24 hours were western blotted for apoptotic markers caspase3 and PARP and anti-apoptotic markers MCL-1, XIAP, and BCL-2. Actin was used as loading control.

Figure 3

TAI-1 inhibits growth of multiple tumor types in xenografted mouse models. Nude mice engrafted with cancer cell lines were treated for 28 days either orally or intravenously as indicated and tumor size measured daily. Huh-7 (A), Colo205 (B), and MDA-MB-231 (C) cells were used. Left panel:% tumor inhibition. Right panel:% body weight.

Figure 4

7-day toxicology study of TAI-1 in mice shows no significant change in body weight, organ weight, and plasma indices. C.B-17 SCID mice (n = 8) were orally administered TAI-1 for 7 days and body weights (A) and organ weights (B) were measured. Liver (C) and kidney (D) plasma indices were determined.

Figure 5

TAI-1 does not inhibit a number of kinases and hERG at below 10 μM. (A) Inhibition of kinases were performed with 10 μM TAI-1 with standard assays. (B) hERG inhibition was determined with 10 μM TAI-1. Results show good cardiac safety of TAI-1.

Figure 6

TAI-1 GI₅₀s correlates with Hec1 protein expression in cancer cell lines. Asynchronously maintained cell lines are lysed and their total protein immunoblotted for expression levels of Hec1. Hec1 protein expression levels are quantitated and expressed in% relative to HeLa expression levels.

Figure 7

Efficient knockdown of RB in cancer cells increases cellular sensitivity to TAI-1. (A) MDA-MB-231 cells which carry wild-type RB were transfected with control siRNA (siControl) or siRNA of RB (siRB) for 24 hours and treated with TAI-1 (starting dose 100 μM, 3x serial dilution), incubated for 48 hours and analyzed for viability with MTS. Cellular sensitivity is expressed in GI₅₀ (nM) and RNA from transfected cells were analyzed for RB RNA level by quantitative real time PCR. SiRB reduced GI₅₀ of compound in cells. (B) Selected cell lines which carry wild type RB (MDA-MB-231, K562, ZR-75-1, T47D, A549, HCT116) or mutated RB (HeLa, as control) were transfected with siRB and treated with TAI-1, incubated for 48 hours and analyzed for viability with MTS. Cellular sensitivity is expressed as% growth inhibition and cell lysates from transfected cells were collected and RB protein levels determined by western blotting. Shown are representative results from at least two independent experiments.

Figure 8

Efficient P53 knockdown in cancer cells increases cellular sensitivity to TAI-1. (A) A549 and HCT116 cells which carry wild-type P53 were transfected with control siRNA (siControl) or P53 siRNA (siP53) for 24 hours and treated with TAI-1 (starting dose 100 μM, 3x serial dilution), incubated for 48 hours and analyzed for viability with MTS. Cellular sensitivity is expressed in GI₅₀ (nM) and RNA from transfected cells were analyzed for P53 RNA level by quantitative real time PCR. SiP53 reduced GI₅₀s of compound in cells. (B) Selected cell lines which carry wild type P53 (A549, HCT116, ZR-75-1, U2OS) or mutated P53 (HeLa, as control) were transfected with siP53, treated with TAI-1 and analyzed for viability with MTS. Cellular sensitivity is expressed as% growth inhibition and cell lysates from transfected cells were collected and P53 protein levels determined by western blotting.

Figure 9

Differential expression of NDC80 (Hec1) and genes associated with NDC80 between subtypes of non-small cell lung cancer. (A) NDC80 (Hec1) (Affymetrix Probeset ID 204162_at) expression between adenocarcinoma and squamous cell carcinoma of lung in three different independent datasets (GSE8894, GSE3141 and GSE37745). The unit of Y axis is logarithm of expression intensity to the base 2. ANOVA was used to compare these two subtypes of NSCLC. (B) One way hierarchical clustering analysis of NDC80 gene and genes associated with NDC80 for subtypes of NSCLC in the same three independent datasets. The results consistently showed up-regulated expression of NDC80 and its closely associated genes (SPC25, NUF2 and Nek2) in squamous cell carcinoma of lung. Green: adenocarcinoma. Yellow: squamous cell carcinoma. The heat map scale is mean ± 2SD.