Background
Drugs that interfere with mitosis are part of the most successful cancer chemotherapeutic compounds currently used in clinical practice [
1]. Development of chemotherapeutic drugs that target the mitotic cycle has focused on inhibition of the mitotic spindle through interactions with microtubules [
1]. Drugs targeting microtubules such as taxanes and vinca alkaloids are effective in a wide variety of cancers, however, the hematopoietic and neurological toxicities as well as development of resistance to this class of drugs severely limit their long term clinical utility [
1,
2]. Novel anti-mitotic agents have been designed to target the mitotic apparatus through non-microtubule mitotic mediators such as mitotic kinases and kinesins [
2].
A novel attractive non-microtubule target is Highly Expressed in Cancer 1 (Hec1), a component of the kinetochore that regulates the spindle checkpoint. Hec1 is of particular interest because of its association with cancer progression [
3‐
5]. Hec1 directly interacts with multiple kinetochore components including Nuf2, Spc25, Zwint-1, and with mitotic kinases Nek2 and Aurora B [
6,
7] and its expression is tightly regulated in both normal cells and transformed cells during the cell cycle [
4,
8]. Rapidly dividing cells express a high level of Hec1, in contrast to very low to undetectable levels of Hec1 in terminally differentiated cells [
3]. Hec1 has been demonstrated to overexpress in various human cancers including the brain, liver, breast, lung, cervical, colorectal and gastric cancers [
3,
9]. From a mechanistic standpoint, targeted inhibition of Hec1 by RNAi or by small molecules effectively blocks tumor growth in animal models [
3,
10]. Therefore, Hec1 emerges as an excellent target for treating cancer clinically.
Small molecules targeting the Hec1/Nek2 pathway was first discovered by Drs. Chen in the laboratory of Dr. W.H. Lee using the inducible reverse yeast two-hybrid screening of a library of ~24,000 compounds [
3]. A series of compounds was designed based on this published initial hit molecule as the starting template to optimize the potency for drug development (Huang
et al., manuscript in preparation). The original template with micromolar
in vitro potency was improved to low nanomolar potency, enabling possible clinical utility of the Hec1-targeted compound. This study explores the features and potential of the improved anticancer agent targeting Hec1, TAI-1, for preclinical development and clinical utility. The
in vitro and
in vivo biological activity, mechanism of action, toxicity and safety, and translational implications are investigated.
Methods
Cell lines
MDA-MB-231, MDA-MB-468, K562, HeLa, MCF7, HCC1954, A549, COLO205, U2OS, Huh-7, U937, HepG2, KG-1, PC3, BT474, MV4-11, RS4;11, MOLM-13, WI-38, HUVEC, RPTEC, and HAoSMC were from Development Center for Biotechnology, New Taipei City, Taiwan; MDA-MB-453, T47D, ZR-75-1, ZR-75-30, MDA-MB-361, Hs578T, NCI-H520, Hep3B, PLC/PRF/5 were from Bioresource Collection and Research Center, Hsinchu, Taiwan. Cell lines were maintained in complete 10% fetal bovine serum (Biowest, Miami, FL, USA or Hyclone, Thermo Scientific, Rockford, IL, USA) and physiologic glucose (1 g/L) in DME (Sigma, St. Louis, MO, USA). Studies conducted using cell lines RPMI8226, MOLT-4, and N87; drug-resistant cell lines MES-SA/Dx5, NCI/ADR-RES, and K562R were from and tested by Xenobiotic Laboratories, Plainsboro, NJ, USA.
In vitropotency assay
Cells were seeded in 96 well plates, incubated for 24 hours, compounds added and incubated for 96 hours. All testing points were tested in triplicate wells. Cell viability was determined by MTS assay using CellTiter 96® Aqueous Non-radioactive Cell Proliferation Assay system (Promega, Madison, WI, USA) according to manufacturer’s instructions with MTS (Promega) and PMS (Sigma, St. Louis, MO). Data retrieved from spectrophotometer (BIO-TEK 340, BIOTEK, VT, USA) were processed in Excel and GraphPad Prism 5 (GraphPad Software, CA, USA) to calculate the concentration exhibiting 50% growth inhibition (GI50). All data represented the results of triplicate experiments.
Immunoblot and co-immunoprecipitation analysis
Western blotting and co-immunoprecipitation were done as described previously [
3]. Primary antibodies used: mouse anti-Nek2 and mouse anti-Mcl-1 (BD Pharmingen, San Diego, CA); rabbit anti-Hec1 (GeneTex, Inc., Irvine, CA); mouse anti-actin (Sigma); mouse anti-P84 and mouse anti-RB (Abcam, Cambridge, MA); rabbit anti-Cleaved Caspase3, rabbit-anti-Cleaved PARP, rabbit anti-XIAP, and mouse anti-P53 (Cell Signaling Technology, Boston, MA); mouse anti-Bcl-2 (Santa Cruz); mouse anti
-α-Tubulin (FITC Conjugate; Sigma).
For co-immunoprecipitation, cells were lysed in buffer (50 mM Tris (pH 7.5), 250 mM NaCl, 5 mM EDTA (pH 8.0), 0.1% Triton X-100, 1 mM PMSF, 50 mM NaF, and protease inhibitor cocktail (Sigma P8340)) for 1 hour then incubated with anti-Nek2 antibody (rabbit, Rockland) or IgG as control (rabbit, Sigma-Aldrich, St. Louis, MO) for 4 hours at 4°C, collected by protein G agarose beads (Amersham) and processed for immunoblotting.
Immunofluorescent staining and microscopy
For quantification of mitotic abnormalities, cells were grown on Lab-Tek® II Chamber Slides, washed with PBS buffer (pH 7.4) before fixation with 4% paraformaldehyde. Following permeabilization with 0.3% Triton X-100, cells were blocked with 5% BSA/PBST and incubated with anti-α-Tubulin antibodies. Then DAPI (4’,6’-diamidino-2-phenylindole) staining was applied and cells were mounted with ProLong® gold antifade (Life Technologies). Images were examined with NIKON 80i microscope at 400× or 1000x magnification and captured with Spot Digital Camera and Spot Advanced Software Package (Diagnostic Instruments, Sterling Heights, MI). The percentage of cells with mitotic abnormalities was calculated by the number of the cells showing the abnormal mitotic figures (including chromosomal misalignment and formation of multipolar spindles) divided by the total number of mitotic cells counted. A minimum of 500 cells from randomly selected fields were scored per condition per experiment.
Mouse xenograft model
The procedure was adapted from published protocol [
3] and were in accordance to the Institutional Animal Care and Use Committee of DCB. C.B-17 SCID mice (6-7 weeks, 21-24 g) (Biolasco, Taipei, Taiwan) were used. Females were used for Colo-205 and Huh-7 while and males were for MDA-MB-231. Cells were injected subcutaneously into the flank in 50% matrigel solution (BD Biosciences, San Jose, CA). 1×10
7, 3×10
6, and 6×10
6 implanted cells/mouse was used for Huh-7, Colo-205, and MDA-MB-231, respectively. Treatment initiated when tumor volume reached 150 mm
3. For Colo-205 and Huh-7, mice were treated with vehicle control (10% DMSO 25% PEG200) per oral PO/BID/28 cycles in total. For Huh-7, a dose increase was incurred on day 4 to increase efficacy. For Colo205, a dose decrease was incurred on day 13 to decrease body weight loss. For MDA-MB-231, mice were treated with vehicle control (5% DMSO, 10% Cremophor, 85% water for Injections (WFI)) per oral PO/BID/28 cycles in total, or TAI-1 formulated in vehicle (20 mg/kg intravenously IV/QDx28 cycles or 150 mg/kg per oral PO/BID/28 cycles in total). Tumor size were measured with digital calipers and volume calculated using the formula (L x W x W)/2, of which L and W represented the length and the width in diameter (mm) of the tumor, respectively. Body weights and tumor growth were measured twice a week. Mean tumor growth inhibition of each treated group was compared with vehicle control and a tumor growth inhibition value calculated using the formula: [1-(T/C) ×100%] (T: treatment group, C: control group tumor volume).
Pilot toxicology study in mice
A sub-acute toxicology study was performed for TAI-1. Female C.B-17 SCID mice (7 weeks old) were used in this study. Mice were divided into four treatment groups: vehicle control (10% DMSO, 25% PEG200, 65% double distilled H2O), test article (in vehicle) at 7.5, 22.5, and 75.0 mg/kg, and all mice were treated twice a day by oral administration for 7 days (n = 8 for each group). Body and organ weights were measured. Blood were collected by cardiac puncture and serum analyzed for complete blood count and biochemical indices.
In vitrokinase assay
Inhibition of kinase activity by test compound was estimated by [33P] labeled radiometric assay. 20 kinase assays (Millipore) were adapted. The kinase reaction was performed according to individual manual with minor modification. In brief, each test compound was evaluated at two concentrations (10 mM and 1 mM) in duplication. The kinase reaction were initiated by enzyme addition, stopped at indicated time by the addition of 3% phosphoric acid, harvested onto a filter plate by using a unifilter harvester (PerkinElmer), and counted by using TopCount (PerkinElmer). The results were the average of duplicate measurements and expressed as percentage inhibition (compound treatment versus DMSO control).
Cardiac toxicology study - hERG binding assay
[3H]Astemizole competitive binding assays are performed to determine the ability of compounds to displace the known radioligand [3H]-astemizole from the hERG potassium channels, following standard protocol with minor modifications. In brief, assays were performed in 200 μl of binding buffer (50 mM HEPES, pH 7.4, 60 mM KCl, and 0.1% BSA) containing 1.5 nM of [3H]astemizole, 3 μg/well of hERG membrane protein (PerkinElmer), and TAI-1 (in 1% DMSO final concentration) at 27°C for 60 min. Nonspecific binding (NSB) was determined in the presence of 10 μM astemizole. IC50 assay for TAI-1 contained 8 concentration points with 10-fold serial dilution in triplicate. Binding was terminated by rapid filtration onto polyethyleneimine-presoaked, buffer-washed UniFilter-96, and GF/C (Perkin Elmer) using a vacuum manifold (Porvair Sciences). Captured radiolabel signal was detected using TopCount NXT (Perkin Elmer). The data were analyzed with nonlinear curve fitting software (PRISM, Graphpad) and IC50 value (defined as the concentration at which 50% of [3H]-astemizole binding is inhibited) was calculated. All results are derived from two independent experiments.
Drug-drug synergy experiments
Interaction (synergy, additive, antagonistic activities) between Hec1 inhibitor TAI-1 and anticancer drugs (sorafenib, doxorubicin, paclitaxel, and topotecan) were evaluated using standard assays. Twenty-four hours after seeding, cells were treated with TAI-1, the other testing drug, or in combination. For combination testing, TAI-1 or the other testing drugs were added to plate in triplicate wells in ratios of GI
50 (GI
50A: GI
50B), and cells are incubated in drug-treated medium for 96 h and cell viability determined by MTS. Synergy was determined by calculating combination index (CI) value with the formula
where CA,X and CB,X are concentrations of drug A and drug B used in combination to achieve x% drug effect. ICx,A and ICx,B are concentrations for single agents to achieve the same effect. All data represent results of triplicate experiments (and data on mean of three separate determinations had variations of less than ±20%).
Gene silencing by siRNA transfection
Cells were seeded onto 96-well plates and transfected with siPort NeoFx transfection method (Ambion, Inc., TX, USA) according to manufacturer’s instructions. Cells were cultured for 24 h and treated with compound. SiRNA from two different sources were used to confirm results. At least two independent experiments are used to determine representative results. Control siRNA (#4390843, Ambion, Inc., Austin, TX, USA or #6568 s, Cell Signaling Technology or sc-37007, Santa Cruz Biotechnology), RB siRNA (Silencer Select ID:s523, Ambion or sc-29468, Santa Cruz Biotechnology), and P53 siRNA (#6231 s, Cell Signaling Technology, or sc-29435, Santa Cruz Biotechnology) were employed. The sequences of these control siRNAs are detailed in the manufacturer websites.
Quantitative real-time RT-PCR
Total RNA was isolated with Quick-RNA miniPrep (Zymo Research, Irvine, CA, USA). Reverse transcription and quantitative real-time PCR was performed on ABI Prism 7500 (PE Applied Biosystems, TX, USA) using the One-Step SYBR ExTaq qRT-PCR kit (Takara, Shiga, Japan) according to manufacturer’s instructions. The following primers were used:
for GAPDH
5′-GGTTTACATGTTCCAATATGATTCCA-3′ (forward), and 5′-ATGGGATTTCCATTGATGACAAG -3′ (reverse);
for RB
5′-GCAGTATGCTTCCACCAGGC-3′ (forward), and 5′-AAGGGCTTCGAGGAATGTGAG-3′ (reverse); and
for P53
5′-GCCCCCAGGGAGCACTA-3′ (forward), and
5′-GGGAGAGGAGCTGGTGTTG-3′ (reverse).
Gene expression in clinical samples–data from databases
NDC80 (Hec1) gene expression data in non-small cell lung cancer (NSCLC) were retrieved from publicly available database (Gene Expression Omnibus-GSE8894, GSE3141 and GSE37745). Gene expression intensities were normalized with quantile normalization. NDC80 expression between adenocarcinoma and squamous carcinoma was compared for all three different datasets. Eight genes known to associate with NDC80 were identified (18, 27). One way hierarchical clustering analysis for adenocarcinoma and squamous carcinoma of NSCLC was conducted by using R package software (
http://www.r-project.org/).
Discussion
This study explored the potential of the improved anticancer agent targeting Hec1 for clinical development and utility. The potency, safety, synergistic effect, markers for response and clinical relevance was evaluated using in vitro, in vivo, and database analysis methods.
Ever since Hec1 was discovered and characterized, the possibility that this may be a good molecular target was discussed. Hec1 is an oncogene that when overexpressed in transgenic mice leads to tumor formation [
5]. The differential expression profile of Hec1 in cancer cells in comparison to normal non-actively dividing cells further supports the suitability of this target for anticancer treatment. The current study shows a small molecule with largely improved potency range enabling the preclinical development of a Hec1 targeted small molecule. The structure-activity relationship is demonstrated for over 200 analogues of the Hec1-targeted small molecule (Huang
et al, manuscript in preparation).
The improved Hec1-targetd small molecule TAI-1 inhibits the growth of a wide spectrum of cancer cell lines
in vitro. Interestingly, a small number of cell lines were resistant to TAI-1, suggesting that there may be changes in signaling pathways that allow cells to bypass Hec1 inhibitor induced cell death. This observation prompted our further exploration of markers for TAI-1 response, which may have clinical implications for personalized therapy. A number of known cellular factors were assessed for their impact on the cellular response to TAI-1. The expression of Hec1, its interacting partner RB [
29], and P53, a tumor suppressor like RB, were evaluated based on possible crosstalk of pathways. The profile in Table
1 shows a possible association of the status of the tumor suppressors with cellular sensitivity to TAI-1. Analysis of the three factors indicate that the participation of RB is nominal (Table
4), however, the
in vitro siRNA studies show that RB may play a role in TAI-1 sensitivity (Figure
7). The impact of RB remains to be clarified in future biomarker studies. In contrast, the combined markers Hec1 and P53 showed a significant impact on cellular sensitivity to TAI-1 (Table
4). In addition, the role of P53 is further supported by the
in vitro siRNA knockdown studies (Figure
8). Although these are very interesting findings, a larger study to allow multivariate analysis will be necessary for more accurate evaluation, but such study is beyond the scope of the current study. Nevertheless, these findings provide a rationale for the building of the parameters for response into future clinical studies for Hec1 inhibitors, in particular TAI-1, and analogues of TAI-1.
In contrast to in vitro cell line studies, the in vivo models demonstrated efficacy but doesn’t reflect the potency from in vitro studies. Administration of drug to animal models, in comparison to cell lines in culture, adds another level of complexity due to possible variability in drug absorption levels due to barriers encountered during oral administration, such as enzymatic degradation, pH sensitivity, drug pumps in the gastrointestinal tract, etc.; hence, the efficacy values between the in vivo models and in vitro models cannot be directly comparable. It is therefore only appropriate to use these preliminary xenograft models to determine efficacy but not to efficacy doses directly to in vitro GI50. Furthermore, better comparison of the efficacy doses between xenograft models should be designed so absorption levels are controlled and formulation of the vehicle for administration is optimized. Note that we are the first to evaluate the oral efficacy of Hec1-targeted inhibitors as an anticancer agent and demonstrate efficacy of the improved Hec1-targeted compound in human liver, colon and breast in vivo tumor models. Even though the great leap in in vitro potency doesn’t correlate well with the in vivo efficacy, this study provides a basis for the pharmaceutical development of a Hec1-targeted small molecule based on the significant improvement in in vitro efficacy, which translates to a clinically applicable oral dosage. The pharmacological parameters, such as oral absorption, and compound solubility remains to be overcome by further modifications to the core structure and exploration of dosing formulations through the efforts of medicinal chemists and formulation experts.
The safety of TAI-1 was evaluated with activity in normal cell lines, hERG inhibition and a pilot toxicity study. The activity in normal cell lines suggests that TAI-1 has high cancer cell specificity and a high therapeutic index. In combination with hERG inhibition assay, the
in vitro evaluation shows that TAI-1 is safe as an anticancer agent with little liability on cardiac toxicity. Furthermore,
in vivo toxicity studies in the same species of mice as the xenograft studies showed no body weight loss and no changes in organ weight and plasma indices. These athymic mice used for
in vivo modeling were good correlation of the toxicity incurred at efficacy doses in the xenograft models, but were unable to show immunosuppression, a common side effect of chemotherapeutics. In rodent with intact thymus, dosing of TAI-1 lead to a dose-dependent decrease of thymus weights and a slight decrease of spleen weights, but did not showed dose-dependent changes in blood indices, including white blood cells, due to TAI-1 (Additional file
2: Figure S1). It should be noted that it is also possible that the lack of body weight loss and hematological effects may not be evident in only 7 days, and toxicity studies dosed for longer period of times may be able to further determine the long term effects of TAI-1. In contrast to the 7-day toxicity study conducted independently of the xenograft studies in SCID mice, xenograft studies seemed to show a modest body weight loss (up to 13.5% at day 7, n = 6) during dosing (Figure
3). Since this effect was not evident in the independently conducted toxicity studies in the same species of mice (0% change at day 7, n=8), the body weight loss is suggested to be nonspecific to the compound. The body weight loss may be related to the tumor burden or different tumor xenograft interactions, since the change varied between models (11.5% for Huh-7 and 13.5% for Colo205 at day 7). The influencing factors of body weight loss in the xenograft models remains unclear, and further parallel designs of xenograft and toxicity studies may help determine the underlying cause.
The translational implications were further explored with studies in multi-drug resistant (MDR) cell lines, synergistic studies, and clinical databases. The activity in MDR cell lines was shown with other Hec1 analogues (Huang et al., manuscript submitted) and is not specific to the Hec1 analogue TAI-1. The activity in MDR cell lines carry important clinical implications and suggests that Hec1-targeted agents may be able to offered as a treatment option to cancer patients who do not respond to currently available anticancer agents, a major clinical advance. A combinatorial approach incorporating anti-cancer drugs targeting different pathway for treatment regimens is often used to improve medical outcomes. The synergistic effects of TAI-1 with commercial anticancer agents suggest that TAI-1 or its analogues may be very easily incorporated to current multi-drug treatment regimens. A small pilot study using clinical database analysis shows that Hec1 expression may correlate with established patient subtypes, which may further aid in the building of the parameters for response in clinical applications. Further studies in the clinical development of Hec-1 inhibitors will determine whether selection based on these subtypes will aid in the identification of patients who are more likely to respond to Hec1-targeted therapy.
Competing interests
LYLH, CCC, KJK, JYNL are employees or consultants of Taivex Therapeutics which owns the rights of this compound. YSL, JJH, JMC, SHC, YJT, PYT, CWL, HSL are employees of Development Center of Biotechnology which collaborated with Taivex Therapeutics and will receive royalty of this compound if successfully approved and marketed.
Authors’ contributions
LYLH carried out the biomarker studies, participated in the design of the cellular, xenograft and toxicology studies, drafted and revised the manuscript. YSL initiated and designed the cell line GI50 screening and mechanistic studies. JJH designed and produced the molecule TAI-1. CCC carried out the studies designed by LYL including cell line GI50 screening, synergy, and the apoptotic blots. JMC designed and participated in the animal studies. YJT carried out the toxicology studies. PYT carried out the xenograft studies. SHH produced TAI-1 for the animal studies. KJK concepted and carried out the clinical sample analysis. CWL carried out western blotting studies for Hec1/Nek2 interaction. HSL carried out the chromosome phenotype studies. JYNL initiated, concepted, and participated in the Hec1/Nek2 inhibitor project and did critical revisions of the manuscript. All authors read and approve the final manuscript.