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Cancer Chemother Pharmacol

DOI 10.1007/s00280-014-2540-7
ORIGINAL ARTICLE
Inhibition of Hec1 as a novel approach for treatment of primary
liver cancer
Lynn YL Huang Chia-chi Chang Ying-Shuan Lee Jiann-Jyh Huang
Shih-Hsien Chuang Jia-Ming Chang Kuo-Jang Kao Gillian MG Lau
Pei-Yi Tsai Chia-wei Liu Her-Sheng Lin Robert G. Gish Johnson YN Lau
Received: 14 January 2014 / Accepted: 11 July 2014
Springer-Verlag Berlin Heidelberg 2014
activity in selected cancer cell lines with doxorubicin,
paclitaxel, and topotecan, but not with sorafenib. TAI-
95 shows excellent potency in a Huh-7 xenograft mouse
model when administered orally. Gene expression analysis
of clinical samples demonstrated increased expression of
Hec1/NDC80 and associated genes (Nek2, SMC1A, and
SMC2) in 27 % of patients, highlighting the potential for
using this therapeutic approach to target patients with high
Hec1 expression.
Conclusion Inhibition of Hec1 using small molecule
approach may represent a promising novel approach for the
treatment of primary liver cancers.
Keywords NDC80 HCC Anticancer drug
Therapeutics Mitosis
Abbreviations
Hec1 Highly expressed in cancer protein 1
HCC Hepatocellular carcinoma
GI
50
Growth inhibition concentration
Introduction
Hepatocellular carcinoma (HCC) is a major cause of cancer
death worldwide [1]. Only 1015 % of HCC patients are
suitable candidates for curative treatment including surgi-
cal resection and liver transplantation. A current treatment
option such as transarterial localized treatment modalities
does not produce long-term clinical results [2]. Further-
more, systemic chemotherapy for HCC has shown incon-
sistent efcacy with side effects [3]. Currently, sorafenib
is the only molecular-targeted agent approved for the treat-
ment of advanced HCC, but the overall efcacy remains
moderate and advanced HCC is still incurable [4]. HCC
Abstract
Purpose Highly expressed in cancer protein 1 (Hec1) is
an oncogene and a promising molecular target for novel
anticancer drugs. The purpose of this study was to evaluate
the potential of a Hec1 inhibitor, TAI-95, as a treatment for
primary liver cancer.
Methods In vitro and in vivo methods were used to test
the activity of TAI-95. Gene expression analysis was used
to evaluate clinical correlation of the target.
Results In vitro growth inhibition results showed that
TAI-95 has excellent potency on a wide range of primary
liver cancer cell lines (hepatoblastoma or hepatocellu-
lar carcinoma) (GI
50
3070 nM), which was superior to
sorafenib and other cytotoxic agents. TAI-95 was rela-
tively inactive in non-cancerous cell lines (GI
50
> 10 M).
TAI-95 disrupts the interaction between Hec1 and Nek2
and leads to degradation of Nek2, chromosomal misalign-
ment, and apoptotic cell death. TAI-95 showed synergistic
Electronic supplementary material The online version of this
article (doi:10.1007/s00280-014-2540-7) contains supplementary
material, which is available to authorized users.
L. Y. Huang (*) C. Chang K.-J. Kao G. M. Lau
R. G. Gish J. Y. Lau
Taivex Therapeutics Corporation, 17th Floor, No. 3, Yuanqu
Street, Nangang District, Taipei City 115, Taiwan, ROC
e-mail: lynnhuang.lh245@gmail.com
Y.-S. Lee J.-J. Huang S.-H. Chuang J.-M. Chang P.-Y. Tsai
C. Liu H.-S. Lin
Development Center for Biotechnology, 101, Lane 169,
Kangning Street, Xizhi District, New Taipei City 22180, Taiwan,
ROC
R. G. Gish
University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093, USA
Cancer Chemother Pharmacol
1 3
is an unmet medical need awaiting novel therapy to be
discovered.
Hec1 is a novel anticancer target that is part of the kine-
tochore that regulates mitotic processes. The kinetochore
links chromosomes to microtubule polymers from the mitotic
spindle to ensure proper chromosome segregation, maintain-
ing genetic integrity [5]. Hec1 is upregulated and recruited
to the kinetochore during cell cycle progression [6], and its
activity is regulated by Nek2 and Aurora B [7]. Disruption of
Hec1 function leads to abnormal mitotic processes resulting
in apoptotic cell death [8]. Hec1 is overexpressed in human
cancers, and its overexpression hyperactivates the mitotic
checkpoint and induces liver carcinoma in vivo [810], high-
lighting its role in liver tumorigenesis. Targeting Hec1 by
RNAi or small molecules effectively blocks tumor growth in
animal models [8, 11, 12]. Therefore, Hec1 emerges as an
excellent target for treating primary liver cancer.
Small molecules targeting the Hec1/Nek2 pathway
were rst identied through yeast two-hybrid screening of
~24,000 compounds by Dr. Wen-hwa Lees group [8]. We
applied rational drug design to the initial hit that was iden-
tied in order to optimize the compound for drug develop-
ment and successfully developed a series of potent small
molecule Hec1 inhibitors that demonstrated good potency
and safety prole ([13, 14] and unpublished observations).
We then further optimized its oral absorption prole to pro-
duce an anticancer compound suitable for further develop-
ment [15]. Since oral administration targets the liver more
effectively, this compound has the potential to provide a
clinical advance for the treatment of liver cancer. This study
aims to investigate the features of TAI-95 to demonstrate its
excellent potential as a novel drug candidate for HCC.
Materials and methods
Cell lines
Huh-7, HepG2 (hepatoblastoma), WI-38, HUVEC,
RPTEC, and AoSMC (Development Center for Biotechnol-
ogy (DCB)), Hep3B and PLC/PRF/5 (Bioresource Collec-
tion and Research Center (Hsinchu, Taiwan)), JHH-1, JHH-
2, JHH-4, and JHH-7 (JCRB Cell Bank), and SNU-423,
SNU-387, SNU-475, SNU-182, SNU-449, and SNU-398
(ATCC) cell lines were maintained in 10 % fetal bovine
serum (Biowest, Miami, FL) with physiologic glucose con-
centration (1 g/L) DME (Sigma, St. Louis, MO) at 37 C
5 % CO
2
.
In vitro drug potency assay
Cells were seeded in 96-well plates and incubated for
24 h. The compounds were then added in triplicate wells
and incubated for 96 h. Cell viability was determined by
MTS assay using CellTiter 96

Aqueous Non-radioactive
Cell Proliferation Assay system (Promega, Madison, WI)
with MTS and PMS (Sigma). Data retrieved from spec-
trophotometer (BIO-TEK 340, BioTeK, Winooski, VT)
were processed in Excel and GraphPad Prism 5 (GraphPad
Software, La Jolla, CA) to calculate relative concentration
for 50 % growth inhibition (GI
50
). Results were generated
from data originating from triplicate experiments. GI
50
s
was also determined for rst-line anticancer drugs pacli-
taxel, doxorubicin, topotecan, irinotecan, gemcitabine, and
sunitinib (Sigma), sorafenib (LC Laboratories, Woburn,
MA), genitib (Selleckchem, Houston, TX), and cisplatin
(Merck, Darmstadt, Germany).
Immunoblot analysis
Western blotting and co-immunoprecipitation were car-
ried out as described previously [8]. Primary antibodies
used Nek2, MCL-1, (BD Pharmingen, San Diego, CA);
Hec1 (GeneTex, Inc., Irvine, CA); actin (Sigma); p84,
IGF2, survivin (Abcam, Cambridge, MA), Caspase3,
PARP, XIAP, Hsp70 (Cell Signaling Technology, Bos-
ton, MA); and BCL-2, Cyclin B1, Cyclin D1, p53 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA). Nek2 anti-
body (Rockland) or IgG control (Sigma) were used for
co-immunoprecipitation.
Immunouorescent staining and microscopy
Immunostaining, image processing, and uorescence-
activated cell sorting (FACS) assays were performed as
detailed previously [8]. Zeiss Axioplan 2 microscope
(equipped with a deconvolution module) or Zeiss LSM-
510 META laser scanning confocal microscope was used to
capture images. For Annexin V detection, FITC Annexin V
Apoptosis Detection Kit I (BD Biosciences) was used.
Drug-drug synergy testing experiments
Interaction between Hec1 inhibitor TAI-95 and antican-
cer drugs was evaluated using standard assays. Twenty-
four hours after seeding, cells were treated with TAI-95,
the other testing drug, or in combination. For combina-
tion testing, TAI-95 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 CellTiter 96

Aque-
ous Non-radioactive Cell Proliferation Assay system
(Promega, Madison, WI) with MTS and PMS (Sigma).
Data retrieved from spectrophotometer (BIO-TEK 340,
BioTeK, Winooski,VT) were processed in Excel. Graph-
Pad Prism 5 (GraphPad Software, La Jolla, CA) was
Cancer Chemother Pharmacol
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used to calculate relative concentration for 50 % growth
inhibition (GI
50
). Synergy was determined by calcu-
lating combination index (CI) value with the formula
CI =

C
A,X
/IC
x,A

C
B,X
/IC
x,B

, where C
A,X
and C
B,X

are concentrations of drug A and drug B used in combina-
tion to achieve x % drug effect. IC
x,A
and IC
x,B
are con-
centrations 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 %).
Mouse xenograft HCC model
The procedure was adapted from published protocol [8].
CB.17 SCID mice (67-week old, Biolasco, Taipei, Tai-
wan) were used, and procedures were performed according
to protocols approved by the Institutional Animal Care and
Use Committee (IACUC) of DCB.
For subcutaneous implantation of Huh-7 (liver cancer
cells), mice were injected subcutaneously in the ank with
1 10
8
cells in 50 % Matrigel solution (BD Biosciences,
San Jose, CA). When tumor volume reaches 150 mm
3
,
mice were divided into four groups and orally adminis-
tered vehicle control (1 % carboxymethyl cellulose (CMC)
(male mice) or 20 % (2-hydroxypropyl)--cyclodextrin
(HPCD) (female mice)), or TAI-95 formulated in vehi-
cle (10, 25, 50 mg/kg or 1, 2.5, 10 mg/kg, respectively)
twice/day for 28 days. Body weight was monitored twice
weekly. Tumor size was measured twice weekly using
digital calipers, and volume of tumor was calculated using
formula (L W W)/2, of which L and W represented
length and width in diameter (mm) of tumor, respectively.
Mean tumor growth inhibition was calculated using for-
mula 1 (T/C) 100 % (T: treatment group; C: control
group tumor volume).
Gene expression of HEC1-associated genes in HCC
patients
Expression of NDC80 and three associated genes in hepa-
tocellular carcinoma were studied [16, 17]. Three HCC
gene expression data sets generated by the same Affym-
etrix U133A microarray platform were selected. Two data
sets were obtained from GEO (GSE6764 [http://www.n
cbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE6764] and
GSE9843 [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi
?acc=GSE9843]) and one from EMBL-EBI (E-TANM-36
[http://www.ebi.ac.uk/arrayexpress/experiments/E-
TABM-36/]). Gene expression of 231 samples in three
data sets was scaled to a global mean of 500 and nor-
malized by quantile normalization. Hierarchical cluster-
ing analysis was conducted using R package software
(http://www.r-project.org/).
Results
TAI-95 blocks proliferation of liver cancer cell lines
at nanomolar potency
To examine in vitro antitumor efcacy of TAI-95 (struc-
ture in Fig. 1a) and to address the heterogeneous nature
of HCC, a total of 13 human liver cancer cell lines were
examined. Their characteristics are summarized in Supple-
mentary Table S1 [1821]. Cancer cell lines were treated
with TAI-95 for 96 h and then analyzed with MTS assay.
Results show that 8 of 13 liver cancer cell lines tested were
insensitive (GI
50
> 10 M) to paclitaxel; 12 of 13 cell
lines needed over 4 M sorafenib for 50 % growth inhi-
bition, indicating that sorafenib is a weak direct antitu-
mor agent; 12 of 13 were insensitive to irinotecan; 13 of
13 were insensitive to genitib and cisplatin; and 11 of 13
were insensitive to sunitinib (Table 1). In contrast, 50 % of
the growth inhibition was achieved at lower than 100 nM
TAI-95 in all liver cancer cell lines tested, demonstrating an
excellent spectrum of anti-liver cancer activities (Table 1).
TAI-95 directly inhibits proliferation of tumor cell and
demonstrated superiority over sorafenib (68>650-fold
difference) in all liver cancer cell lines tested. This growth
inhibition prole in a wide spectrum of liver cancer cells
suggests that TAI-95 is not inuenced by tumor and patient
factors related to the origin of the tumor cell lines, includ-
ing HBV status, expression of alpha-fetoprotein, CEA, and
IGF-II overexpression, and ethnic origin of patients from
which these cells were derived.
To determine the selectivity of TAI-95, we studied
the effect of TAI-95 on normal cell lines. TAI-95 was
not active (GI
50
> 10 M) in relatively normal cell lines,
including human broblasts (WI-38), human umbilical
vein endothelial cells (HUVEC), renal proximal tubule
epithelial cells (RPTEC), and human aortic smooth mus-
cle cells (AoSMC), demonstrating a 1,000 times difference
in potency between cancer cell lines and normal cell lines
(Table 1), preliminarily implicating a relatively high thera-
peutic index of TAI-95, at least in the representative tested
normal cell lines organ of origin.
TAI-95 targets the Hec1/Nek2 pathway
To conrm the mechanism of action of TAI-95, co-immu-
noprecipitation and Western blotting assays were per-
formed. In Huh-7 cells treated with TAI-95, Hec1 failed
to co-immunoprecipitate with Nek2, indicating that the
Hec1/Nek2 complex was disrupted by the treatment with
TAI-95 (Fig. 1b). Since previous study also showed that
disruption of the interaction of Hec1 with Nek2 with Hec1
inhibitors leads to degradation of Nek2 protein [8, 12], we
detected the Nek2 protein levels after TAI-95 treatment.
Cancer Chemother Pharmacol
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The overall cellular Nek2 protein level was signicantly
reduced in a dose- and time-dependent manner after TAI-
95 treatment in Huh-7, with little change in Hec1 (Fig. 1c,
d, respectively). Similar results were noted in PLC/PRF/5,
Hep3B, SNU-449, and JHH-4 cell lines (Supplementary
Fig. S1a).
Chromosomal and spindle abnormalities are common to
cells defective in Hec1 or Nek2 [22]. To test whether TAI-
95 treatment generates similar cellular phenotypes, we used
Huh-7 to perform biological studies. The mitotic popula-
tion with chromosomal misalignment increased with TAI-
95 treatment, triggering aberrant spindles such as deformed
or multipolar spindles (Fig. 2a). Furthermore, this mani-
festation was both time- and dose-dependent (Fig. 2a and
Supplementary Fig. S1b). Direct binding assays for Hec1 is
being set up and would provide further support for TAI-95
binding to Hec1, but the observations provided here sug-
gest that TAI-95 targets the Hec1/Nek2 pathway, leading to
the disruption of Hec1/Nek2 interaction, induction of Nek2
degradation, and misalignment of chromosomes.
TAI-95 treatment leads to apoptotic cell death
To determine the type of cell death induced by TAI-95,
we observed the phenotypic changes and detected vari-
ous apoptotic markers in TAI-95-treated liver cancer cells.
TAI-95-treated cells showed cell shrinkage (Fig. 2b left
panel) and chromatin condensation (Fig. 2b right panel),
which are hallmarks of apoptosis. To further characterize
the TAI-95-induced cell death pathway, apoptotic markers
Fig. 1 TAI-95 targets
Hec1/Nek2 pathway. a Structure
of the candidate compound. b
Huh-7 cells treated with DMSO
or 1 M TAI-95 for 3 h were
immunoprecipitated by Nek2
antibody to detect co-immuno-
precipitated Hec1. Control IgG
was used as control antibody
for immunoprecipitation. c, d
DMSO or 1 M TAI-95-treated
Huh-7 cells were immunoblot-
ted for Hec1 and Nek2
Cancer Chemother Pharmacol
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were detected by immunouorescent staining and Western
blotting. The appearance of Annexin V, an inner membrane
leaet marker that appears in early apoptotic cells, and PI,
a marker for late apoptosis, demonstrated that the cells
treated with TAI-95 showed progression to apoptotic cell
death (Fig. 2c). Furthermore, cleavage of apoptotic mark-
ers (PARP and caspase3) and degradation of anti-apoptotic
markers (MCL-1) indicated that TAI-95 activated the apop-
totic pathway (Fig. 3a). No change in XIAP levels was
observed, but BCL-2 levels were decreased (Supplemen-
tary Fig. S2a and S2b).
TAI-95 leads to downregulation of cancer-related proteins
Upregulated and mutated protein expression often leads to
drug resistance of cancer to anticancer agents. To further
determine the potential of TAI-95 in the treatment of liver
cancer, levels of pro-survival and cell cycle proteins [23
26] were detected by Western blotting in TAI-95-treated
cells. TAI-95 leads to degradation of the anti-apoptotic
protein Mcl-1, cyclin D1, IGF2, Hsp70, survivin, and p53
(Fig. 3c). These results were also seen in PLC/PRF/5 and
partially demonstrated in Hep3B (Supplementary Fig.
S3ac).
TAI-95 does not show consistent synergistic effect
with other cytotoxic agents
For the treatment of solid cancer tumors, a combination of
anticancer drugs rather than a single drug is often used. We
investigated the combined effects of TAI-95 with sorafenib,
doxorubicin, paclitaxel, and topotecan using a synergy
assay. A combination index was calculated with the GI
50
s
determined by the assay and used to determine whether
positive effects were present with combination treatment.
No consistent results across the spectrum of cell lines were
observed (Table 2). However, in Huh-7 cells, TAI-95 was
synergistic with doxorubicin, paclitaxel, and topotecan but
not with sorafenib. TAI-95 also was synergistic with doxo-
rubicin in JHH-7 and with paclitaxel in JHH-4 and JHH-
7. TAI-95 was not synergistic with sorafenib in any of the
liver cancer cell lines tested.
TAI-95 monotherapy blocks in vivo Huh-7 tumor growth
To examine the in vivo antitumor efcacy of TAI-95, a nude
mouse model harboring human liver cancer cell line Huh-
7-derived tumor xenograft was used. Oral administration
of 10, 25, and 50 mg/kg of TAI-95 signicantly inhibited
tumor growth (Fig. 4a left panel), while no body weight
loss relative to the vehicle control was noted (Fig. 4a right
panel).
Preclinical clinical implications relevant to TAI-95 target
Hec1
We used publicly available database to identify a
Hec1 gene cluster in liver cancer. All four genes (H
HEC11/NDC80, NEK2, SMC1A, and SMC2) selected
for study showed wide ranges of expression in patients
Table 1 GI
50
of TAI-95 in human liver cancer lines
N.D. not determined
(nM) Sorafenib Doxorubicin Paclitaxel Topo-tecan Irinotecan Genitib Gemcitabine Cisplatin Sunitinib TAI-95
Huh-7 4934 109.28 30.03 71.27 >10,000 >10,000 >10,000 >10,000 >10,000 34.98
Hep3B 3995 154.55 9.19 403.63 >10,000 >10,000 4.56 >10,000 >10,000 58.54
PLC/PRF/5 4379 75.94 5.52 558.74 >10,000 >10,000 12.28 >10,000 >10,000 31.66
HepG2 7794 1532 >10,000 594.85 32236 >10,000 >10,000 >10,000 >10,000 67.4
SNU-182 4046 363.93 >10,000 687.26 >10,000 >10,000 >10,000 >50,000 >10,000 45.43
SNU-387 >10,000 1060 >10,000 1390 >10,000 >10,000 >50,000 >50,000 8828 32.72
SNU-423 6334 833.12 >10,000 576.88 >10,000 >10,000 >10,000 >50,000 >10,000 24.30
SNU-449 4909 >10,000 >10,000 >10,000 >10,000 >10,000 >50,000 >10,000 6279 40.30
SNU-475 >10,000 >10,000 >10,000 >10,000 >10,000 >10,000 >10,000 >50,000 >10,000 15.38
JHH-1 >10,000 1007 >10,000 329.55 >10,000 >10,000 >10,000 >50,000 >10,000 41.90
JHH-2 9022 696.28 >10,000 1371 >10,000 >10,000 >10,000 >50,000 >10,000 45.51
JHH-4 5684 257.03 146.03 50.02 8208 >10,000 11.44 >10,000 >10,000 70.76
JHH-7 7445 466.93 7.00 1652 >10,000 >10,000 >10,000 >10,000 >10,000 65.34
WI-38 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. >10,000
HUVEC N.D. 198 N.D. N.D. N.D. N.D. N.D. N.D. N.D. >10,000
RPTEC N.D. 24003,400 N.D. N.D. N.D. N.D. N.D. N.D. N.D. >10,000
AoSMC N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. >10,000
Cancer Chemother Pharmacol
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of HCC (Supplementary Table S2). Clustering analysis
showed proportional co-expression of these four genes in
231 hepatocellular carcinoma samples (Fig. 4b). Samples
with HEC1 expression above mean (Supplementary Table
S2) and higher expression of all four genes are highlighted
with red bar below in Fig. 4b (n = 63). The results showed
a tight association between HEC1 (NDC80) and NEK2 and
showed that around 27 % of hepatocellular carcinoma has
increased expression of HEC1-associated genes.
Discussion
This study demonstrated the potential of the novel
Hec1/Nek2 inhibitor as novel approach to treating primary
liver cancer. TAI-95 has excellent potency in the growth
inhibition of a wide spectrum of primary liver cancer cells
as well as excellent oral efcacy in the inhibition of tumor
growth in vivo. The disruption of the interaction of Hec1
and Nek2 by TAI-95 leads to the activation of apoptotic
pathway and eventually cell death. Furthermore, gene
expression analysis of clinical HCC patient samples dem-
onstrated the possible percentage and subtypes of HCC
patients that may be sensitive to Hec1-targeted anticancer
therapy.
TAI-95 is a rst-in-class anticancer compound that tar-
gets Hec1, a regulatory kinetochore protein with well-
dened signaling pathways, oncogenic properties in vivo,
ability to be inhibited by small molecules, and specic
expression patterns in fast dividing as well as cancerous
cells [8, 9, 27]. This study showed that TAI-95 has excel-
lent potency in vitro and in vivo. HCC comprises clinically
Fig. 2 TAI-95 targets
Hec1/Nek2 pathway and
leads to apoptotic phenotype.
a DMSO or TAI-95-treated
Huh-7 were stained with tubulin
antibody(microtubules) and
DAPI(DNA), and metaphase
cells (>100) were counted
for presence of chromosomal
misalignment and expressed
as % counted. b Cells treated
with DMSO or TAI-95(1 M)
for 24 h were photographed
directly (left panel) or stained
with DAPI (right panel) to
visualize condensed nuclear
chromosomal globules in late-
stage apoptotic cells (arrows). c
DMSO or TAI-95-treated Huh-7
were stained with Annexin
V and PI to detect membrane
changes
Cancer Chemother Pharmacol
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Fig. 3 TAI-95 leads to activa-
tion of apoptotic pathway. ac
DMSO, TAI-95, or sorafenib
(SOR)-treated Huh-7 were
immunoblotted for cleaved
PARP, cleaved caspase3, Mcl-1,
cyclinB1, cyclin D1, IGF2,
Hsp70, survivin, and p53. Actin
or p84 was used as loading
control
Cancer Chemother Pharmacol
1 3
chemo-resistant tumors, demonstrated by low response
rates across available anticancer agents [28]. The multi-
ple etiological factors involved in the nature of the disease
compromise the effective treatment of liver cancer, result-
ing in drug resistance [29]. In this study, TAI-95 was active
against all liver cancer cells irrespective of the HBV sta-
tus or the expression of AFP, while other anticancer drugs
showed poor spectrum of potency. It is likely that the
altered cell death signaling pathways in selected cell lines
have a negative impact on the activity of other rst-line
anticancer drugs. It is possible that TAI-95 is able to bypass
the resistance and survival mechanisms of liver cancer cell
lines through a yet unknown mechanism and induces severe
cell death, but this remains to be conrmed.
Although the combination of TAI-95 and sorafenib did
not show any synergistic effect, the fact that direct anti-
proliferative effect of sorafenib is very weak (600-fold
differences) may not allow synergistic effect to be demon-
strated using our in vitro cellular approach. It is reasonable
to postulate that the combination of the anti-neoangiogen-
esis effect of sorafenib and anti-proliferative and apoptotic
effect of TAI-95 may be clinically synergistic in patients
with HCC. The direct tumor-inhibition activity of TAI-95
on clinical tumors comprised of diverse genetic background
in contrast to in vivo single cell line xenograft models, and
whether there are any synergistic effect clinically remains
to be tested.
Study shows that the balance of pro-apoptotic and anti-
apoptotic proteins is important for the sensitivity of cells to
cell death. Genetic alterations in HCC lead to imbalance in
the pro-apoptotic and anti-apoptotic proteins of the BCL-2
family, of which BCL-X
L
and MCL-1 are overexpressed
[30]. A shift of the apoptosis/anti-apoptosis balance, such
as by downregulation of an essential anti-apoptotic protein
Table 2 Combination index of TAI-95 with anticancer agents
Combination index = 1 means additive, <1 means synergy, >1 means no synergy
N.D., not determined due to resistance (GI
50
> 10 M) to the corresponding drugs
Huh-7 PLC/PRF/5 HepG2 Hep3B SNU-182 SNU-423 JHH-1 JHH-4 JHH-7
Sorafenib 1.28 1.34 1.94 1.42 1.10 1.20 N.D. 1.09 1.35
Doxorubicin 0.76 2.35 3.00 1.66 1.03 1.77 1.47 2.02 0.18
Paclitaxel 0.65 1.33 N.D. 1.61 N.D. N.D. N.D. 0.30 0.62
Topotecan 0.48 2.47 2.83 1.54 1.23 3.73 1.44 1.38 1.44
Fig. 4 TAI-95 inhibits liver
tumor growth and is expressed
in clinical HCC samples. a
Nude mice bearing Huh-7 liver
cancer xenograft were dosed
orally BID with indicated vehi-
cle, and tumor size was meas-
ured. *: P < 0.05; **: P < 0.01,
by two-tailed t test. mpk, mg/
kg. b Hierarchical clustering
analysis of HCC samples and
HEC1 gene cluster. Samples
with HEC1 expression above
mean and higher expression of
all four genes are highlighted
with red bar below (n = 63).
These samples account for 27 %
of HCC samples
Cancer Chemother Pharmacol
1 3
such as MCL-1, may lead to cell sensitivity to cell death. In
this study, we show that TAI-95 leads to the degradation of
various pro-survival and cell cycle proteins, some of which
is associated with resistance to drugs, such as paclitaxel,
doxorubicin, and sorafenib [3133]. Anti-apoptotic and
survival proteins MCL-1, cyclin D1, survivin, and p53 have
all been implicated as regulators of liver cancer cell death
[2326]. The potency of TAI-95 on a wide spectrum of can-
cer cell lines may be associated with the ability to shift the
apoptosis/anti-apoptosis balance; however, this remains to
be elucidated by future studies. Nevertheless, these obser-
vations indicate that the inhibition of Hec1/Nek2 by TAI-
95 may serve as a good approach to the killing of chemo-
resistant HCC cancers.
Personalized medicine matching patients to appro-
priate treatment regimens can greatly increase efcacy
while decreasing adverse effects [34]. Clustering analy-
sis of clinical databases of HCC showed a low percent-
age of clinical HCC with increased Hec1 expression.
This was unexpected since Hec1 is an integral part of
the kinetochore complex that is highly correlated with
increased proliferation in cancer cells. There are two pos-
sible explanations for the low percentage shown by the
database analysis. Firstly, some HCC may utilize alterna-
tive growth pathways and bypass the Hec1 pathway. Sec-
ondly, given the short half-life of mRNA of these genes,
the samples collected might be in suboptimal condition
leading to under-representation of the actual gene expres-
sion levels. Nevertheless, these data showed that Hec1 and
its related genes are at least over-expressed in a propor-
tion of patients with HCC. The sensitivity of cancer cell
lines to Hec1/Nek2 inhibitors is positively correlated with
the expression level of Hec1 (Huang et al., in press), and
higher HEC1 expression in 27 % of HCC patients suggests
that not all HCC patients may respond equally well to the
Hec1 inhibitor. The current analysis of clinical liver cancer
subtypes provides a basis for selection of patients that are
more likely to be responsive to Hec1-targeted treatment.
The predictability of response to Hec1-targeted treatment
according to Hec1-associated gene expression remains to
be seen; however, our results suggest such consideration
for Hec1 or related gene expression may be an important
factor in the clinical design of personalized Hec1 targets
treatment of HCC.
In conclusion, the potency, efcacy, and translational
prole of TAI-95 demonstrate that the inhibition of the
Hec1 oncogene is a promising novel approach to the treat-
ment of primary liver cancers.
Acknowledgments We thank our team members at the Develop-
ment Center for Biotechnology, Jeffrey Chia-Lin Wang, Drs. Chi-feng
Chang, Jui-Lien Huang, Horace Loh, and Ms. Lihyan Lee, and Mr.
Kuo-ming Yu for their unfailing support. This research was funded by
Taivex Therapeutics and the Development Center for Biotechnology
grant issued by the Ministry of Economic Affairs of the Republic of
China (102-EC-17-A-01-05-0739, to Chia-ling Wang).
Conflict of interest LYLH, CCC, KJK, GML, JYNL, and RGG are
either employees or consultants of Taivex Therapeutics, which owns
the rights of this compound. YSL, JJH, SHC, JMC, YJT, PYT, CWL,
and 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.
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