1

KIFC1 regulates ZWINT to promote tumor progression and spheroid
formation in colorectal cancer

Running title: Role of KIFC1 and ZWINT in CRC

Shintaro Akabane1,2, Naohide Oue1, Yohei Sekino3, Ryuichi Asai4, Pham QuocThang1, Daiki Taniyama1, Kazuhiro Sentani1, Masashi Yukawa5, Takashi Toda5,
Ken-ichi Kimura6, Hiroyuki Egi2, Wataru Shimizu2, Hideki Ohdan2, Wataru
Yasui1*

1

Department of Molecular Pathology, Graduate School of Biomedical and Health

Sciences, Hiroshima University
2

Department of Gastroenterological and Transplant Surgery, Graduate School of

Biomedical and Health Sciences, Hiroshima University
3

Department of Urology, Graduate School of Biomedical and Health Sciences,

Hiroshima University
4

Department of Surgical Oncology, Graduate School of Medicine, Gifu University

2

5

Hiroshima Research Center for Healthy Aging, Department of Molecular

Biotechnology, Graduate School of Integrated Sciences for Life, Hiroshima University
6

Chemical Biology Laboratory, Graduate School of Arts and Sciences, Iwate University*Corresponding author:
Wataru Yasui, MD, PhD
Department of Molecular Pathology, Graduate School of Biomedical and Health
Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551,
Japan.
E-mail address: wyasui@hiroshima-u.ac.jp
Phone number: +81 82 257 5145, Fax number: +81 82 257 5149

Abbreviated words:
ALDH1, aldehyde dehydrogenase 1; CI, confidence interval; CRC, colorectal cancer;
CSCs, cancer stem cells; EGFR, epidermal growth factor receptor; KIFC1, kinesin
family member C1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; KAA, kolavenic acid analog; siRNA, small interfering RNA; TCGA, The
Cancer Genome Atlas; ZWINT, ZW10 interacting kinetochore protein

3

ABSTRACT
Colorectal cancer (CRC) is the second leading cause of cancer-related mortality
worldwide. Kinesin Family Member C1 (KIFC1) has been proposed as a promisingtherapeutic target due to its pivotal role in centrosome clustering to mediate cancer cell
progression. This study aimed to analyze the expression and biological function of
KIFC1 in CRC. Immunohistochemically, 67 (52%) of 129 CRC cases were positive for
KIFC1 and statistically associated with poorer overall survival. KIFC1 siRNAtransfected cells demonstrated lower cell proliferation as compared to the negative
control cells. A specific KIFC1 inhibitor, kolavenic acid analog (KAA) drasticallyinhibited CRC cell proliferation. Microarray analysis revealed that KAA-treated CRC
cells presented reduced ZW10 interacting kinetochore protein (ZWINT) expression ascompared to control cells. Immunohistochemical analysis demonstrated that 61 (47%)
of 129 CRC cases were positive for ZWINT and ZWINT expression was significantly
correlated with KIFC1 expression. ZWINT-positive cases exhibited significantly worse
overall survival. KIFC1 siRNA-transfected cells showed reduced ZWINT expression
while ZWINT siRNA-transfected cells decreased cell proliferation. Both KIFC1 andZWINT knockdown cells attenuated spheroid formation ability. This study provides
new insights into KIFC1 regulating ZWINT in CRC progression and its potential as atherapeutic target.

Keywords: colorectal cancer, KIFC1, kolavenic acid analog, ZWINT

4Introduction
Colorectal cancer (CRC) is estimated to have more than 1.8 million new cases (ranks
third) and 881,000 deaths (ranks second) worldwide according to the Global Cancer
Statistics in 2018.1 The survival rate of patients with early-stage CRC is relatively high;however, approximately 60 % of the cases are found in the later stage.2 The five-year
survival rate of stage IV patients is estimated to drop as low as 12.5 %.3 Therefore, there
is an urgent need for determining a novel molecular target for CRC treatment strategies.
In recent years, cancer has been regarded as a stem cell disease.4 In solid tumors,
cancer stem cells (CSCs) have been characterized by specific cell surface markers, suchas CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1).5 Generally, in the processof spheroid colony formation, cells are cultured in non-attachment dishes with serumfree media, which effectively enriches CSCs.6 Previously, we examined the gene
expression profile of the spheroid colonies formed by gastric cancer (GC) cells using
microarray analysis and found that kinesin family member C1 (KIFC1) expression was
upregulated in GC spheroid colonies.7 The kinesin superfamily proteins share a motor
domain and participate in several biological functions.8 Reportedly, KIFC1 plays apivotal role in centrosome clustering in cancer cells and is related to the pathogenesis ofseveral cancer types.9–11 Generally, cancer cells with centrosome amplification lead to
apoptosis through multipolar mitosis; however, centrosome clustering could prevent cell
death causing the formation of pseudo-bipolar structures.12 Although in our previous

5study, we reported that KIFC1 is overexpressed in gastric, esophageal, and prostatecancers and this overexpression is probably involved in cancer stemness,7,13,14 the roleof KIFC1 in CRC is yet to be investigated. Therefore, we aimed to investigate KIFC1
expression using immunohistochemistry, examine the association between KIFC1
expression and clinicopathologic characteristics, and analyze the biological function of
KIFC1 with small interfering RNA (siRNA) on cell growth and spheroid formation in
CRC cells in the present study.
Previously, kolavenic acid analog (KAA), a natural compound, has been identified as a
KIFC1-specific inhibitor through the screening method using KIFC1-overproducing
fission yeast cells.15,16 In the current study, we aimed to elucidate the effect of thiscompound on CRC cell proliferation. Further, we conducted microarray analysis toreveal the change in expression of several genes, including KIFC1, investigated thelevel of ZW10 interacting kinetochore protein (ZWINT) expression and elucidated thebiological role of ZWINT in CRC.

Materials and methods
Tissue samples and cell lines
In a retrospective study design, 129 surgically resected tumors were collected from
CRC patients underwent curative resection between 2006 and 2010 at Hiroshima

6

University Hospital (Hiroshima, Japan). Because written informed consent was not
obtained, for strict privacy protection, all of the identifying information associated withthe samples was removed before the analysis. This study was approved by the EthicalCommittee for Human Genome Research of Hiroshima University, Hiroshima, Japan(no. IRINHI66). We previously demonstrated that KIFC1 induces resistance tochemotherapy,14 we considered the preoperative treatment could affect the expression
status of KIFC1. Therefore, patients who received preoperative radiotherapy or
chemotherapy were excluded from the study. Further, operative mortality was defined as
death within 30 days of patients leaving the hospital, wherein these patients were also
excluded from the analysis. Postoperative follow-up appointments were scheduled every
3 months during the first 2 years and every 6 months over the next 3 years and yearly
thereafter. Chest X-ray, computed tomography scan, and serum tumor markers wereassessed at every follow-up visit. Patients were followed by their respective physicians
until the patients’ death or the date of the last documented contact. Human colon cancerderived cell lines, DLD-1, LoVo, WiDr, and COLO201 were purchased from theJapanese Collection of Research Bioresources Cell Bank (Osaka, Japan). All cell lines
were maintained in RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd., Tokyo,
Japan) containing 10 % fetal bovine serum (BioWhittaker, Walkersville, MD, USA) in ahumidified atmosphere of 5 % CO2 and 95 % air at 37 °C.

Immunohistochemistry

7

One or two representative tumor blocks, including the tumor center, invasive front, andtumor-associated non-neoplastic mucosa, were examined from each sample byimmunohistochemistry. In case of large, late-stage tumors, two different sections were
examined to include representative areas of the tumor center as well as of the lateral anddeep tumor invasive front. Immunohistochemical analysis was performed using Dako
EnVision+ Mouse Peroxidase Detection System (Dako Cytomation, Carpinteria, CA,
USA). Antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0)
for 30 min. Peroxidase activity was blocked with 3 % H2O2-methanol for 10 min andnon-specific antibody binding sites were blocked by incubating the sections with normal
goat serum (Dako Cytomation) for 20 min. Further, the sections were incubated withmouse monoclonal anti-KIFC1 (1:100; Abnova), anti‐CD44 (1:100, clone DF1485;
Novocastra), anti-ALDH1 (1:400, clone 44/ALDH1; BD Biosciences), and rabbit
polyclonal anti‐ZWINT (1:50, clone HPA005757; Sigma-Aldrich) antibodies for 1 h atroom temperature, followed by incubation with EnVision+ anti-mouse peroxidase for 1
h. For color reaction, sections were incubated with the DAB substrate-chromogen
solution (Dako Cytomation) for 10 min. Sections were counterstained with 0.1 %hematoxylin. Negative controls were created by the omission of the primary antibody.
Expressions of KIFC1, CD44, and ALDH1 were evaluated as positive when >10 % of
tumor cells were stained. ZWINT immunostaining was evaluated and scored using
German immunoreactive score as previously described.18 Intensity score was assessed
as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Percentage of positive tumor
cells were scored as 0 (< 5 %), 1 (5 %–25 %), 2 (25 %–50 %), 3 (50 %–75 %), or 4 (>

875 %). Staining intensity and staining area scores were multiplied as the final
immunoreactive score of ZWINT expression. Subsequently, a final score ≥ 6 wasdefined as ZWINT-positive and a score ≤ 4 was defined as ZWINT-negative. Using
these definitions, two surgical pathologists (NO and KS), without knowledge of theclinicopathological parameters or the patients’ outcomes, independently reviewed
immunoreactivity in each specimen. Interobserver differences were resolved byconsensus review at a double-headed microscope after independent review.

RNAi and cell growth assay
siRNA oligonucleotides targeting KIFC1, ZWINT, and negative control werepurchased from Invitrogen (Carlsbad, CA, USA). We used two independent KIFC1 andZWINT siRNA oligonucleotide sequences. Transfection was performed using
Lipofectamine RNAiMAX (Invitrogen) as previously described.20 Briefly, 60 pmol ofsiRNA and 10 μL of Lipofectamine RNAiMAX were mixed in 1 mL of RPMI-1640
medium (10 nmol/L final siRNA concentration). After 20 min of incubation, the mixture
was added to cells, and then cells were plated in culture dishes. Forty-eight hours after
transfection, cells were analyzed as described further. To examine cell growth, MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed. The
transfected cells were seeded at a density of 4,000 cells/well in 96-well plates. Cell
growth was monitored at 1, 2, and 4 days.

9

Spheroid colony formation
For the generation of spheroids, 1,000 cells were seeded in a 6-well ultra-low
attachment plate (Corning, Corning, NY, USA). Cells were grown in mTeSR medium(StemCell Technologies Inc.). The plates were incubated at 37 °C in an incubator with a
5 % CO2 atmosphere for 15 days. Spheroid number and size were determined andcounted under a microscope.

Treatment by KIFC1 inhibitor, and gene expression analysis
Kolavenic acid analog (KAA) was previously isolated from Solidago altissima andidentified as a KIFC1-specific inhibitor.15,16 It was dissolved in DMSO as a stock
solution and stored at -20℃ before use. DLD-1 and WiDr cells were treated with KAA
followed by MTT assays performed at 1, 2, and 4 days. Further, microarray analysis was
performed using the Affymetrix GeneChip® manual (Affymetrix Inc., Santa Clara, CA,USA), as previously described.21 RNA was isolated from DLD-1 cells treated with KAA(25 μM) for 48 h and control cells. Gene alteration was calculated by fold change.

Statistical methods
Associations between clinicopathological parameters and KIFC1 (ZWINT) expression
were analyzed by Fisher's exact test. We used Kaplan–Meier method to assess survival
estimates and log-rank test to compare KIFC1 (ZWINT)-positive and negative cases.

10

The association between clinical covariates and cancer-specific mortality was evaluatedusing univariate and multivariate Cox regression analyses. Hazard ratio (HR) with its
95 % confidence interval (CI) was estimated accordingly. Differences between groups
in the MTT and spheroid formation assays were evaluated by the Student t-test. Cell
growth and spheroid formation assays involved three independent experiments; mean ±SD was calculated for each of these experiments. SPSS, ver. 24 (IBM Corp, Armonk,
NY, USA) was used for these analyses, with a significant level set at p < 0.05.

Quantitative reverse transcription PCR (qRT-PCR), Western blot, Gene expression
data from The Cancer Genome Atlas (TCGA), Wound healing assay, Conventional
adenoma tissue samples
These methods are described in detail in the Supporting Information.

Results
Expression of KIFC1 in CRC tissue samples
We first investigated the KIFC1 mRNA expression in 14 types of normal tissue and 28
CRC tissue samples by qRT-PCR. Clinicopathologic characteristics of these 28 CRC
tissue samples are shown in Supporting Table 2. The gastrointestinal tissues (stomach,11

small intestine, and colon) showed a relatively high KIFC1 expression among thenormal tissue samples, whereas the CRC tissue samples showed remarkably higher
KIFC1 expression as compared to the normal colon tissue. (Fig. 1a). We next performed
immunohistochemical analysis in 129 surgically resected CRC tissue samples. Weak or
no staining of KIFC1 was observed in the epithelial and stromal cells of non-neoplasticcolonic mucosa. Conversely, CRC tissue samples showed robust, more extensive
staining mainly in the nucleus (Fig. 1b). Many CRC tissue samples demonstrated
heterogeneous KIFC1 staining wherein the proportion of KIFC1-stained CRC cells
ranged from 0 % to 40 %. Upregulation of KIFC1 in the invasive front was not found.
We regarded KIFC1 immunostaining as positive when more than 10 % of the tumor
cells were stained. In total, 67 (52 %) of 129 CRC cases were found positive for KIFC1.
We further investigated the relationship between KIFC1 and CSC markers byimmunohistochemistry and found that CD44 and ALDH1 were mainly stained in themembrane and the cytoplasm of the CRC cells, respectively (Fig. 1c). KIFC1-positive
CRC cases were significantly correlated with CD44-positive (p = 0.011) and ALDH1positive (p = 0.009) cases (Table 1). We then examined the relationship between KIFC1
expression and clinicopathological characteristics (Table 2). KIFC1-positive cases weresignificantly associated with N and M stages and showed significantly poorer overall
survival (Fig. 1d). In TCGA database, short-term survival (1 to 80 months) in CRC
patients with high KIFC1 expression was significantly decreased as compared to thosewith low KIFC1 expression (Fig. 1e), which was consistent with our analysis. Further,
multivariate analysis showed that KIFC1 expression was an independent prognostic

12

factor for overall survival in CRC patients (Table 3). These results indicate that KIFC1
expression is associated with CRC progression and could be involved in CRC stemness.

KIFC1 inhibition affected cell growth, EGFR-related pathway, and spheroid
formation in CRC cells
We next examined the biological role of KIFC1 using CRC cell lines. Western blot
analysis revealed various levels of KIFC1 expression in CRC cells (Fig. 2a). We
selected DLD-1 and WiDr cells that showed high KIFC1 expression for further
experiments. We found that the KIFC1 expression was substantially suppressed bytreatment with KIFC1-specific siRNAs (siRNA1 and siRNA2) by immunoblotting (Fig.
2b). MTT assays performed after siRNA transfection demonstrated that KIFC1 siRNAtransfected cells showed significantly reduced cell growth as compared to the negative
control cells (Fig. 2c). It has been reported that epidermal growth factor receptor(EGFR) activates the Ras-mitogen-activated protein kinase kinase-Erk andAkt-phosphatidylinositol- 4,5-bisphosphate 3-kinase signaling pathways which lead tocancer cell proliferation and survival in colorectal cancer.22,23 To investigate the KIFC1‐
induced activation of EGFR-related pathway in CRC, we analyzed the levels of Akt andErk phosphorylation in CRC cells with KIFC1 inhibition. Consequently, we found thatthe levels of phosphorylated Akt and Erk (pAkt and pErk, respectively) were reduced in
KIFC1 siRNA‐transfected cells (Fig. 2b). We next analyzed the association between
KIFC1 expression and spheroid formation ability in CRC cells. We observed that the

13number and size of spheroids were significantly reduced in KIFC1 siRNA-transfected
cells as compared to the control cells (Fig. 2d). These results suggest that KIFC1 isinvolved in cell growth through the EGFR-related pathway and is required for spheroid
formation in CRC.

KIFC1-specific inhibitor blocked cell proliferation and altered gene expression
DLD-1 and WiDr cells were treated with KAA at various concentrations for 4 days.
MTT assays showed that KAA inhibited cell proliferation in a dose-dependent manner(Fig. 3a). Wound healing assay also revealed that KAA inhibited CRC migration in adose-dependent manner (Supporting Figure 1). Microarray analysis to evaluate the genealterations by KAA revealed multiple genes that were significantly decreased by KIFC1
inhibition (Supporting Table 3). We examined the expressions of these genes in 10 CRC
tissue samples and calculated the ratio of mRNA expression levels between cancer
tissue (T) and corresponding non-neoplastic mucosa (N). From a total of seven genes,
ZWINT was overexpressed (T/N ratio of > 2) in 6 (60 %) of the 10 cases (Supporting
Figure 2). Therefore, we next investigated the expression of ZWINT in CRC.
Immunohistochemical analysis showed that ZWINT expression was observed mainly inthe cytoplasm of cancer cells and KIFC1-positive regions (Fig. 3b). In total, 61 (47 %)of 129 CRC cases were positive for ZWINT. Moreover, ZWINT-positive cases weresignificantly associated with KIFC1-positive cases (Table 2) and higher TNM stages(Supporting Table 4). We additionally investigated the expressions of KIFC1 and14

ZWINT in colorectal adenoma specimens and found that these molecules were scarcely
detected (images are not shown), suggesting that KIFC1 and ZWINT would not be
involved in the conventional adenoma-carcinoma pathway (Supporting Figure 3).
ZWINT-positive cases showed poorer overall survival (Fig. 3c), whereas the short-term
survival of CRC patients with high ZWINT expression was not statistically significant in
TCGA database (data not shown). We further analyzed the expressions of these genes
using TCGA database (n = 512) and found that ZWINT expression was significantly
correlated with KIFC1 expression (Fig. 3d). Further, multivariate analysis showed thatZWINT expression was an independent prognostic factor for overall survival (Table 3).
We next confirmed the protein reduction in CRC cells after KAA treatment (25 μM) andfound that KIFC1 and ZWINT were downregulated in a time-dependent manner (Fig.
3e). We also demonstrated that the expression of ZWINT was reduced in KIFC1
siRNA‐transfected cells (Fig. 3f). These results indicate that the KIFC1 inhibitor could
block CRC cell proliferation and regulate ZWINT expression that affects the prognosis
of CRC.

ZWINT inhibition affected cell growth, EGFR-related pathway, and spheroid
formation
We next investigated the effect of ZWINT inhibition on CRC cell functions. We
confirmed reduced expression of ZWINT by the transfection of ZWINT-specificsiRNAs (siRNA1 and siRNA2) (Fig. 4a). MTT assay showed decreased cell growth15ability in ZWINT siRNA-transfected cells (Fig. 4b). Levels of pAkt and pErk werereduced in ZWINT siRNA‐transfected cells as compared to the negative control cells,
whereas the expression of KFC1 was not affected by ZWINT knockdown (Fig. 4a).
Further, the number and the size of spheroids of ZWINT siRNA-transfected cells weresignificantly smaller than those of the negative control cells (Fig. 4c). These results
indicate that ZWINT could play a crucial role as a downstream gene of KIFC1 in CRC
progression.

Discussion
In the present study, we found a novel association between KIFC1 and CRC. We
demonstrated that 52 % of our study cohort were positive for KIFC1 in the
immunohistochemical analysis and KIFC1 expression was significantly associated withlater-stage disease and poorer prognosis. In the current situation, the survival of CRC
patients with the metastatic disease still remains low in spite of the development ofchemotherapies.24 Further, lymph node-positive CRC patients who receive conventional
oxaliplatin-based adjuvant chemotherapy suffer from severe sensory neurotoxicity.25
KIFC1 plays an important role in centrosome clustering for cancer cell survival,12,26
whereas KIFC1 is dispensable in normal cells that have only one pair of centrosomes.27
Both the qRT-PCR and immunohistochemical analyses presented that the expression of
KIFC1 in CRC tissue samples was much higher than that of the normal tissue samples.

16

We further demonstrated that KAA (KIFC1-specific inhibitor) could drastically inhibit
CRC cell proliferation and migration. These results indicate that KIFC1-inhibiting
strategy would entail an aggressive effect on CRC with fewer adverse events and that
immunohistochemical analysis of KIFC1 could be available in selecting patients who
benefit from KIFC1-targeted therapy.
Microarray analysis demonstrated that ZWINT was downregulated in KAA-treatedcells. ZWINT is previously identified as an essential component of chromosome
motility and spindle checkpoint control.28,29 ZWINT overexpression has been associatedwith poor prognosis in hepatocellular carcinoma and lung cancer,18,30 whereas theexpression and biological role of ZWINT in CRC has not been investigated. In our
immunohistochemical analysis, we found that the positivities between KIFC1 andZWINT were significantly correlated and both expressions were independent prognostic
predictors in CRC patients. In the cell functional analysis, KIFC1 and ZWINT
inhibition significantly reduced cell proliferation. We further confirmed that theexpressions of pAkt and pErk were suppressed by KIFC1 and ZWINT knockdown,
indicating that these genes might be involved in the activation of the EGFR-related
pathway in CRC progression. Taken together, these results suggest that KIFC1 andZWINT could be involved in the malignant behavior of CRC.
CSC-targeting strategies are of great importance because CSCs are associated withseveral malignant features of CRC.31 We previously demonstrated that KIFC1 could
affect cancer stemness.7,13,14 In this study, we showed that KIFC1-positive cases showeda significant correlation with CD44 and ALDH1-positive CRC cases. CSCs have been

17

reported to be a minor population (< 5 %) of cancer cells;32 however,immunohistochemical analysis showed the various proportions of KIFC1-stained CRC
cells, which ranged from 0 % to 40 %, implying that KIFC1 would not be a specificmarker for colorectal CSCs. We also found that KIFC1 and ZWINT knockdown further
reduced the spheroid formation ability of CRC cells, indicating that these genes could
be involved in CRC stemness. Reportedly, KIFC1 and ZWINT are key genes associatedwith cancer stemness in other cancer types as demonstrated by in silico analyses.33,34
however, the underlying mechanism between CRC stemness and these genes remain
unknown. Further investigation is warranted to clarify the biological implications of
KIFC1 and ZWINT in colorectal CSCs.
There are certain limitations associated with this study. Firstly, this study was a
retrospective one involving a small size of clinical samples for the
immunohistochemical analyses. A prospective study with a larger number of CRC
patients is needed to verify our study findings. Secondly, the mechanism of direct orindirect interaction between KIFC1 and ZWINT is still unclear that demands further
analysis in this regard.
In summary, this study demonstrated that KIFC1 regulated ZWINT to facilitate CRC
progression and could induce an unfavorable prognosis among CRC patients. Further, invitro experiments showed that KIFC1 and ZWINT affected cell proliferation via EGFRrelated pathway and spheroid formation. We found that the KIFC1 inhibitor
dramatically reduced CRC cell proliferation, indicating that KIFC1 could be a
promising therapeutic target for CRC.

18

Acknowledgments
The authors are grateful to Mr. Shinichi Norimura for his excellent technical assistanceand advice. This work was supported by Grants-in-Aid for Scientific Research and for
Challenging Exploratory Research from the Japan Society for the Promotion of Scienceand the Takeda Science Foundation.

Disclosure statement
None declared.

Author Contributions
SA (first author), NO and WS (corresponding author) conceptualized and designed thestudy. SA performed data collection and analyses. YS, RA, PQT, DT, and KS assisted
the molecular technologies used. MY, TT and KK isolated the chemical substance usedin the experiment. HE, WS, and HO performed the surgical care and provided clinicalsamples. SA drafted the manuscript. NO provided inputs regarding manuscript
preparation. WS contributed to manuscript preparation. All authors have read andapproved the final manuscript.

19

References
1.

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for
36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394-424.

2.

Favoriti P, Carbone G, Greco M, Pirozzi F, Pirozzi REM, Corcione F.
Worldwide burden of colorectal cancer: a review. Updates Surg. 2016; 68: 7-11.

3.

DeSantis CE, Lin CC, Mariotto AB, et al. Cancer treatment and survivorshipstatistics, 2014. CA Cancer J Clin. 2014; 64: 252-71.

4.

Bessède E, Dubus P, Mégraud F, Varon C. Helicobacter pylori infection and stem
cells at the origin of gastric cancer. Oncogene. 2015; 34: 2547-55.

5.

Wakamatsu Y, Sakamoto N, Oo HZ, et al. Expression of cancer stem cellmarkers ALDH1, CD44 and CD133 in primary tumor and lymph node metastasisof gastric cancer. Pathol Int. 2012; 62: 112-9.

6.

Takaishi S, Okumura T, Wang TC. Gastric cancer stem cells. J Clin Oncol. 2008;
26: 2876-82.

7.

Oue N, Mukai S, Imai T, et al. Induction of KIFC1 expression in gastric cancer
spheroids. Oncol Rep. 2016; 36: 349-55.

8.

Liu X, Gong H, Huang K. Oncogenic role of kinesin proteins and targeting
kinesin therapy. Cancer Sci. 2013; 104: 651-56.

20

9.

Pannu V, Rida PCG, Ogden A, et al. HSET overexpression fuels tumor
progression via centrosome clustering-independent mechanisms in breast cancer
patients. Oncotarget. 2015; 6: 6076-91.

10.

Han J, Wang F, Lan Y, et al. KIFC1 regulated by miR-532-3p promotes
epithelial-to-mesenchymal transition and metastasis of hepatocellular carcinoma
via gankyrin/AKT signaling. Oncogene. 2019; 38: 406-20.

11.

Li G, Chong T, Yang J, Li H, Chen H. Kinesin motor protein KIFC1 is a target
protein of mir-338-3p and is associated with poor prognosis and progression ofrenal cell carcinoma. Oncol Res. 2018; 27: 125-37.

12.

Cosenza MR, Krämer A. Centrosome amplification, chromosomal instability andcancer: mechanistic, clinical and therapeutic issues. Chromosome Res. 2016; 24:
105-26.

13.

Imai T, Oue N, Yamamoto Y, et al. Overexpression of KIFC1 and its association
with spheroid formation in esophageal squamous cell carcinoma. Pathol Res
Pract. 2017; 213: 1388-93.

14.

Sekino Y, Oue N, Shigematsu Y, et al. KIFC1 induces resistance to docetaxel
and is associated with survival of patients with prostate cancer. Urol Oncol Semin
Orig Investig. 2017; 35: 31.e13-31.e20.

15.

Yukawa M, Yamauchi T, Kurisawa N, Ahmed S, Kimura K, Toda T. Fission
yeast cells overproducing HSET/KIFC1 provides a useful tool for identification

21

and evaluation of human kinesin-14 inhibitors. Fungal Genet Biol. 2018; 116:
33-41.
16.

Kurisawa N, Yukawa M, Koshino H, Onodera T, Toda T, Kimura K. Kolavenic
acid analog restores growth in HSET-overproducing fission yeast cells andmultipolar mitosis in MDA-MB-231 human cells. Bioorganic Med Chem. 2020;
28: 115154.

17.

Kondo T, Oue N, Yoshida K, et al. Expression of POT1 is Associated with
Tumor Stage and Telomere Length in Gastric Carcinoma. Cancer Res. 2004; 64:
523-9 LP-529.

18.

Ying H, Xu Z, Chen M, Zhou S, Liang X, Cai X. Overexpression of Zwint
predicts poor prognosis and promotes the proliferation of hepatocellular
carcinoma by regulating cell-cycle-related proteins. Onco Targets Ther. 2018;
11: 689-702.

19.

Yasui W, Ayhan A, Kitadai Y, et al. Increased expression of p34cdc2 and its
kinase activity in human gastric and colonic carcinomas. Int J Cancer. 1993; 53:
36-41.

20.

Sakamoto N, Oue N, Sentani K, et al. Liver-intestine cadherin induction by
epidermal growth factor receptor is associated with intestinal differentiation ofgastric cancer. Cancer Sci. 2012; 103: 1744-50.

22

21.

Fujita T, Chiwaki F, Takahashi RU, et al. Identification and characterization of
CXCR4-positive gastric cancer stem cells. PLoS One. 2015; 10: 1-19.

22.

Li S, Schmitz KR, Jeffrey PD, Wiltzius JJW, Kussie P, Ferguson KM. Structural
basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer
Cell. 2005; 7: 301-11.

23.

Fang JY RB. The MAPK signalling pathways and colorectal cancer. Lancet
Oncol. 2005; 6: 322-27.

24.

Cimino SK, Eng C. Up-and-coming experimental drug options for metastatic
colorectal cancer. J Exp Pharmacol. 2020; 12: 475-85.

25.

Grothey A, Sobrero AF, Shields AF, et al. Duration of adjuvant chemotherapy
for stage III colon cancer. N Engl J Med. 2018; 378: 1177-88.

26.

Kim N, Song K. KIFC1 Is Essential for Bipolar Spindle Formation and Genomic
Stability in the Primary Human Fibroblast IMR-90 Cell. Cell Struct Funct. 2013;
38: 21-30.

27.

Xiao Y-X, Yang W-X. KIFC1: a promising chemotherapy target for cancer
treatment? Oncotarget. 2016; 7: 48656-70

28.

Wang H, Hu X, Ding X, et al. Human Zwint-1 specifies localization of Zeste
White 10 to kinetochores and is essential for mitotic checkpoint signaling. J Biol
Chem. 2004; 279: 54590-8.

23

29.

Lin YT, Chen Y, Wu G, Lee WH. Hec1 sequentially recruits Zwint-1 and ZW10
to kinetochores for faithful chromosome segregation and spindle checkpoint
control. Oncogene. 2006; 25: 6901-14.

30.

Yuan W, Xie S, Wang M, et al. Bioinformatic analysis of prognostic value of
ZW10 interacting protein in lung cancer. Onco Targets Ther. 2018; 11: 1683-95.

31.

Todaro M, Francipane MG, Medema JP, Stassi G. Colon Cancer Stem Cells:
Promise of Targeted Therapy. Gastroenterology. 2010; 138: 2151-62.

32.

Clarke MF, Al-Hajj M, Morrison SJ, Wicha MS, Benito-Hernandez A.
Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci.
2003; 100: 3983-88.

33.

Pan S, Zhan Y, Chen X, Wu B, Liu B. Identification of Biomarkers forControlling Cancer Stem Cell Characteristics in Bladder Cancer by Network
Analysis of Transcriptome Data Stemness Indices. Front Oncol. 2019; 9: 1-11.

34.

Liu D, Xu X, Wen J, et al. Integrated Genome-Wide Analysis of Gene
Expression and DNA Copy Number Variations Highlights Stem Cell-Related
Pathways in Small Cell Esophageal Carcinoma. Stem Cells Int. 2018; 2018: 1-9.

24

Figure legends
Figure 1 Expression of KIFC1 in CRC tissue samples. (a) qRT-PCR analysis of KIFC1
in 14 kinds of normal tissues and 28 CRC tissue samples. The units are arbitrary, andKIFC1 expression was standardized against 1.0 μg of total RNA from normal colon
tissue as 1.0. (b) Immunohistochemical staining of KIFC1 in colon adenocarcinoma.
Left panel, original magnification, ×40. Right panel, a high-magnification image of the
field designated by a box in the left panel. Original magnification, ×400. (c)Immunohistochemical analysis of KIFC1, CD44, and ALDH1. Original magnification,
×100. Right-upper panel, a high-magnification image of the field designated by a blackbox. Original magnification, ×400. (d) Kaplan–Meier plot of overall survival of CRC
patients. (e) Kaplan–Meier plot of short-term survival (1 to 80 months) of CRC patientsin TCGA database.

Figure 2 Functional analysis of KIFC1 in CRC cells. (a) Western blot analysis of
KIFC1 in 4 CRC cell lines. (b) Western blot analysis of KIFC1, Akt, phospho-Akt(pAkt), Erk 1/2 and phospho-Erk1/2 (pErk1/2) in lysates of DLD-1 and WiDr cells
transfected with KIFC1 siRNA or negative control siRNA. β-actin was used as aloading control. (c) Effects of KIFC1 knockdown on DLD-1 and WiDr cell growth. Cell
growth was assessed spectrometrically by MTT assay at 1, 2, and 4 days after seeding.
Bars and error bars indicate mean and S.D., respectively, of three independent
experiments. (d) Number and size of spheroids formed by DLD-1 and WiDr cells

25

transfected with KIFC1 or negative control siRNAs. Bars and error bars indicate mean
and S.D., respectively, of three independent experiments.

Figure 3 Cytotoxic effect of KIFC1 inhibitor and analysis of ZWINT expression. (a)KAA inhibited CRC growth in a dose-dependent manner. Cell growth was assessed
spectrometrically by MTT assay at 1, 2, and 4 days after seeding. Bars and error bars
indicate mean and S.D., respectively, of three independent experiments. NS, notsignificant. (b) Immunohistochemical staining of KIFC1 and ZWINT in colon
adenocarcinoma. Original magnification, ×40. Right-lower panel, a high-magnification
image of the field designated by a black box. Original magnification, ×400. (c) Kaplan–
Meier plot of overall survival of CRC patients. (d) Correlation between KIFC1 andZWINT expressions in colorectal adenocarcinoma samples in TCGA gene-level
expression data (n = 512). (e) KIFC1 and ZWINT expressions reduced by KIFC1
inhibitor in a time-dependent manner. (f) ZWINT expression downregulated in KIFC1
siRNA‐transfected cells. β-actin was used as a loading control.

Figure 4 Effect of ZWINT inhibition on the biological functions of CRC cells. (a)Western blot analysis of ZWINT, KIFC1, Akt, pAkt, Erk1/2, and pErk1/2 in lysates of
CRC cells transfected with ZWINT siRNA or negative control siRNA. β-actin was used
as a loading control. (b) ZWINT knockdown inhibited CRC cell growth. Cell growth
was assessed spectrometrically by MTT assay at 1, 2, and 4 days after seeding. Bars and26

error bars indicate mean and S.D., respectively, of three independent experiments. (c)Number and size of spheroids formed by CRC cells transfected with ZWINT or
negative control siRNA. Bars and error bars indicate mean and S.D., respectively, ofthree independent experiments.

List of supplementary material
Supplementary Material and methods Quantitative reverse transcription PCR (qRTPCR), Western blot, Gene expression data from The Cancer Genome Atlas (TCGA),
Wound healing assay, Conventional adenoma tissue samples

Supporting Table 1 Primer sequences used for qRT-PCR
Supporting Table 2 Clinicopathologic characteristics of 28 patients with CRC
Supporting Table 3 List of genes downregulated by the KIFC1-specific inhibitor,
kolavenic acid analog
Supporting Table 4 Correlation between ZWINT expression and clinicopathologic
characteristics

27

Supporting Figure 1 Representative images and wound contraction percentage of awound-healing assay in DLD-1 (a) and WiDr (b) cells treated with KAA. Bars and error
bars indicate mean and S.D., respectively, of three independent experiments. NS, notsignificant.
Supporting Figure 2 Comparison of mRNA expression levels of genes downregulatedby the KIFC1 inhibitor (kolavenic acid analog) in CRC tissue samples. Fold changes
between CRC tissue (T) and corresponding non-neoplastic mucosa (N) aredemonstrated.
Supporting Figure 3 Summary of immunohistochemical staining of KIFC1 andZWINT in conventional colon adenomas. The graph indicates the number of cases withstaining status. CALG, conventional adenoma low grade; CAHG, conventional
adenoma high grade.

28

29

30

31

32

Table 1 Correlation between KIFC1 expression and cancer stem cell markers
KIFC1 expression
Positivep-value*

Negative

CD44

0.011
Positive41 (63 %)24

Negative26 (41 %)

38

ALDH1

0.009

Positive37 (65 %)20

Negative30 (42 %)42* Chi-square test

33

Table 2 Relationship between KIFC1 expression and clinicopathologic characteristics
KIFC1 expressionAge ≤ 60 > 60Sex
 Men
 WomenTumor location
Right colon
Left colon
Histological classification
Well/moderate
Poor/mucinous

Positive

Negative26 (62 %)
41 (47 %)

16
46

0.115

0.487
22 (48 %)
45 (54 %)

24
38
0.502

18 (47 %)
49 (54 %)

20
42
0.822

62 (52 %)
5 (56 %)

58
4

T classification
T1/T2
T3/T4

17 (42 %)
50 (57 %)24
38

N classification
N0
N1/N2/N3

25 (40 %)42 (63 %)37
25

M classification
M0
M1

44 (44 %)
23 (82 %)

57
5

Stage
Stage I/II
Stage III/IV

21 (36 %)46 (65 %)37
25

ZWINT expression
Negative
Positive* Chi-square test

p-value*

0.104

0.011

< 0.001

0.001< 0.00120 (29 %)
47 (77 %)

48
14

34

Table 3 Univariate and multivariate Cox regression analysis of overall survival

Age
 ≤60
 >60

Univariate analysis
HR
95 % CI
p-value
0.870
1 (Ref.)
0.946
0.486–1.840

Sex
 Men
 Women1 (Ref.)
0.756

Tumor location
Right colon
Left colon

1 (Ref.)
0.572

Histological classification
Well/moderate
Poor/mucinous

1 (Ref.)
2.73

Stage
Stage I/II
Stage III/IV

1 (Ref.)12.617 3.867–41.170

KIFC1 expression
Negative
Positive
ZWINT expression
Negative
Positive

Multivariate analysis
HR
95 % CI
p-value

0.427
0.380–1.506
0.100
0.294–1.113
0.058
0.965–7.721< 0.001

0.002
1 (Ref.)
6.997

2.083–23.508< 0.001
1 (Ref.)9.318 3.293–26.369

0.029
1 (Ref.)
3.642

1.142–11.609< 0.001
1 (Ref.)
8.912 3.468–22.906

HR, hazard ratio; CI, confidence interval

0.035
1 (Ref.)
3.139

1.087–9.068

35

Supplementary Material and methods
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and 1
μg of total RNA was converted to cDNA with First Strand cDNA Synthesis Kit(Amersham Biosciences Corp., Piscataway, NJ, USA). qPCR was performed using
SYBR Green PCR Core Reagents Kit (Applied Biosystems, Foster City, CA, USA) andthe emission intensity was detected on ABI PRISM 7700 Sequence Detection System(Applied Biosystems) as described previously.17 β-actin (ACTB gene)-specific PCR
products amplified from the same RNA samples served as internal control. Primer
sequences used in this analysis are summarized in Supporting Table 1.

Western blot
Cells were lysed as previously described.19 The lysates (40 μg) were solubilized in
Laemmli sample buffer by boiling and then subjected to 10 % SDS-PAGE. Following
electrophoresis, the bands were electrotransferred onto a nitrocellulose membrane thatwas further incubated with primary antibodies against KIFC1 and ZWINT. Peroxidaseconjugated anti-mouse and anti-rabbit IgG were used in the secondary reaction.
Immunocomplexes were visualized with ECL Western Blot Detection system(Amersham Biosciences Corp.). β-actin (Sigma, St. Louis, MO, USA) was used as theloading control.

36

Gene expression data from The Cancer Genome Atlas (TCGA)
For analyzing gene expression data, TCGA colon adenocarcinoma gene-level
expression data (n = 439 samples, version 2016) were downloaded from OncoLnc(http://www.oncolnc.org/). In the Kaplan–Meier analysis, we used receiver operating
characteristic (ROC) curve analysis to define the cut-off point for the KIFC1 expressionthat correlated with the prognosis. KIFC1 expression was considered high if theexpression score was above the cut-off value and low if the score was equal to or below
the cut-off value. For analyzing the correlation between KIFC1 and ZWINT expressions,
TCGA colon adenocarcinoma gene-level expression data (n = 512 samples, version2019) were downloaded from UCSC Xena hub (http://xena.ucsc.edu) as log2 values of
the RSEM normalized counts.

Wound healing assay
To carry out the wound healing assay, the treated cells were plated onto collagen̺
coated coverslips. The monolayer cells were then scratched manually with a plastic
pipette tip, and after being washed with PBS, the wounded monolayers of the cells wereallowed to heal in RPMI 1640 containing 10% FBS. Wound healing was measured bywound contraction percentage and captured at 0, 24, 48, or 60 hours after scratching.

37

Conventional adenoma tissue samples
Twenty primary colorectal adenoma samples were collected from patients who
underwent endoscopic or surgical resection at Hiroshima University Hospital or its
affiliated hospitals. The distribution of colorectal adenomas was as follows:
conventional adenoma low grade (CALG), n = 10; conventional adenoma high grade(CAHG), n = 10.

Supporting Table 1 Primer sequences used for qRT-PCR

GeneForward primer

Reverse primer

KIFC1

GACGCCCTGCTTCATCTG

CCAGGTCCACAAGACTGAGG

FAM111B

TGGACAGACTCTCCGGTTCT TCTGCTTCATGACAGTATCCTTG

CEP128GGATCTCTGCAGGACCGTGTA TTCTCAGGCACAGACTCCAGA

METTL7A

GCATTGCAGAGAACCGACAC AAATAGAAAGCCCCTCCCGGT

ARHGEF39 GTCTCCTCAAGGTGACAGCC AACCACCGTCAATGAGCACTERN2TGCTCATTGGACACCACGAG CGGCTGTTGCTCCAAGAAGAACSM3GGTTCAGGGCTGCTCTTCTAZWINT

AGGTTTCTGCAGAGGGTAAGC ACAGCCTTGAAGGACACACCCGCTGAAAACACTTGGCATGA

38

Supporting Table 2 Clinicopathologic characteristics of 28 patients with CRC
Number of cases
Age, median (range)

28
68 (43-93)

Sex (%)Men

14 (50%)Women

14 (50%)Tumor location (%)
Right colon

16 (57%)

Left colon

12 (43%)Differentiation (%)
Well/moderate

22 (79%)Poor/mucinous

6 (21%)

pT classification (%)
T1/T2

9 (32%)T3/T4

19 (68%)pN classification (%)
N0

13 (46%)

N1/N2/N3

15 (54%)pM classification (%)
M0

25 (89%)M1

3 (11%)

pStage (%)
Stage I/II

13 (46%)

Stage III/IV

15 (54%)

39

Supporting Table 3 List of genes downregulated by the KIFC1 inhibitor, kolavenic
acid analog
Gene symbol

Gene name

Fold change

FAM111B

family with sequence similarity 111, member B

0.137738139

CEP128centrosomal protein 128 kDa

0.153893052

METTL7A

methyltransferase like 7A

0.156041319

ARHGEF39

Rho guanine nucleotide exchange factor 39

0.167240944

ERN2

endoplasmic reticulum to nucleus signaling 2

0.211686328

ACSM3

acyl-CoA synthetase medium-chain family member 3 0.251738888

ZWINT

ZW10 interacting kinetochore protein

0.267943366

KIFC1

kinesin family member C1

0.332171454

40

Supporting Table 4 Correlation between ZWINT expression and clinicopathologic
characteristics
ZWINT expression
Positive
NegativeAge≤ 60
 > 600.421
22 (52 %)
39 (45 %)

20
48

Sex
Men
Women

44 (53 %)
17 (37 %)39
29

Tumor location
Right colon
Left colon

20 (53 %)
41 (45.1 %)18
50

Histological classification
Well/moderate
Poor/mucinous

55 (46 %)
6 (67 %)

65
3

pT classification
T1/T2
T3/T4

10 (24 %)
51 (58 %)31
37pN classification
N0
N1/N2/N3

17 (27 %)
44 (66 %)45
23

pM classification
M0
M1
pStageStage I/II
Stage III/IV*
Chi-square test

p-value*

0.080

0.432

0.227

< 0.001< 0.001

0.001
40 (40 %)
21 (75 %)61
7<0.001

15 (26 %)46 (65 %)

43
25

41

(a)

(b)

Supporting Figure 1 Representative images and wound contraction percentage of awound-healing assay in DLD-1 (a) and WiDr (b) cells treated with KAA. Bars and error
bars indicate mean and S.D., respectively, of three independent experiments. NS, notsignificant.

42

2
0

10
8
6
4
2
0

ARHGEF39

12
10
8
6
4
2
0

1 2 3 4 5 6 7 8 9 10
0

ERN2

1 2 3 4 5 6 7 8 9 100

Fold change (T/N)

12
Fold change (T/N)

10
1
8
6
4
2
0

1 2 3 4 5 6 7 8 9 100

F ld changeFoldh g (T/N)
(
)

12
10
1
8
6
4
2
0

Fold change (T/N)

Fold change (T/N)
Fold change (T/N)(T/N

1 2 3 4 5 6 7 8 9 100

F ld change
Fold
h
(T/N)

4

METTL7A

12
1

1 2 3 4 5 6 7 8 9 10

ACSM3

1
12
10
1
8
6
4
2
0

F ld change
Fold
h
(T/N)

6

CEP128
Fold change (T/N)

8

F ld changeFoldh g (T/N)
(
)

10
1

12

Fold change (T/N)

FAM111B
Fold change (T/N)

Fold change (T/N)
Fold change (T/N)

112

1 2 3 4 5 6 7 8 9 10

ZWINT

10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 10

Supporting Figure 2 Comparison of mRNA expression levels of genes downregulatedby the KIFC1 inhibitor (kolavenic acid analog) in CRC tissue samples. Fold changes
between CRC tissue (T) and corresponding non-neoplastic mucosa (N) aredemonstrated.

43

ZWINT

KIFC1
10

Negative

Positive

Negative

Weak

10

8

8

6

6

4

4

2

p = 0.606

0
CALG

CAHG

2

p = 0.531

0
CALG

CAHG

Supporting Figure 3 Summary of immunohistochemical staining of KIFC1 andZWINT in conventional colon adenomas. The graph indicates the number of cases withstaining status. CALG, conventional adenoma low grade; CAHG, conventional
adenoma high grade.