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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, and
KIFC1 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 black
box. 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 patients
in 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 a
loading 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, not
significant. (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 and
ZWINT 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, of
three 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 a
wound-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, not
significant.
Supporting Figure 2 Comparison of mRNA expression levels of genes downregulated
by the KIFC1 inhibitor (kolavenic acid analog) in CRC tissue samples. Fold changes
between CRC tissue (T) and corresponding non-neoplastic mucosa (N) are
demonstrated.
Supporting Figure 3 Summary of immunohistochemical staining of KIFC1 and
ZWINT in conventional colon adenomas. The graph indicates the number of cases with
staining 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
Positive
p-value*
Negative
CD44
0.011
Positive
41 (63 %)
24
Negative
26 (41 %)
38
ALDH1
0.009
Positive
37 (65 %)
20
Negative
30 (42 %)
42
* Chi-square
test
33
Table 2 Relationship between KIFC1 expression and clinicopathologic characteristics
KIFC1 expression
Age
≤ 60
> 60
Sex
Men
Women
Tumor location
Right colon
Left colon
Histological classification
Well/moderate
Poor/mucinous
Positive
Negative
26 (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
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
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
< 0.001
20 (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
Women
1 (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) and
the 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 that
was 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 the
loading 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 expression
that correlated with the prognosis. KIFC1 expression was considered high if the
expression 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, version
2019) 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 were
allowed to heal in RPMI 1640 containing 10% FBS. Wound healing was measured by
wound 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
Gene
Forward primer
Reverse primer
KIFC1
GACGCCCTGCTTCATCTG
CCAGGTCCACAAGACTGAGG
FAM111B
TGGACAGACTCTCCGGTTCT TCTGCTTCATGACAGTATCCTTG
CEP128
GGATCTCTGCAGGACCGTGTA TTCTCAGGCACAGACTCCAGA
METTL7A
GCATTGCAGAGAACCGACAC AAATAGAAAGCCCCTCCCGGT
ARHGEF39 GTCTCCTCAAGGTGACAGCC AACCACCGTCAATGAGCACT
ERN2
TGCTCATTGGACACCACGAG CGGCTGTTGCTCCAAGAAGA
ACSM3
GGTTCAGGGCTGCTCTTCTA
ZWINT
AGGTTTCTGCAGAGGGTAAGC ACAGCCTTGAAGGACACACC
CGCTGAAAACACTTGGCATGA
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
CEP128
centrosomal 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
Negative
Age
≤ 60
> 60
Sex
Men
Women
0.421
22 (52 %)
39 (45 %)
20
48
0.080
44 (53 %)
17 (37 %)
39
29
20 (53 %)
41 (45.1 %)
18
50
Histological classification
Well/moderate
Poor/mucinous
55 (46 %)
6 (67 %)
65
pT classification
T1/T2
T3/T4
10 (24 %)
51 (58 %)
31
37
pN classification
N0
N1/N2/N3
17 (27 %)
44 (66 %)
45
23
Tumor location
Right colon
Left colon
pM classification
M0
M1
pStage
Stage I/II
Stage III/IV
Chi-square test
p-value*
0.432
0.227
< 0.001
< 0.001
0.001
40 (40 %)
21 (75 %)
61
<0.001
15 (26 %)
46 (65 %)
43
25
41
(a)
(b)
Supporting Figure 1 Representative images and wound contraction percentage of a
wound-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, not
significant.
42
10
ARHGEF39
12
10
1 2 3 4 5 6 7 8 9 10
ERN2
1 2 3 4 5 6 7 8 9 10
Fold change (T/N)
12
Fold change (T/N)
10
1 2 3 4 5 6 7 8 9 10
F ld change
Fold
h g (T/N)
12
10
Fold change (T/N)
Fold change (T/N)
Fold change (T/N)
(T/N
1 2 3 4 5 6 7 8 9 10
F ld change
Fold
(T/N)
METTL7A
12
1 2 3 4 5 6 7 8 9 10
ACSM3
12
10
F ld change
Fold
(T/N)
CEP128
Fold change (T/N)
F ld change
Fold
h g (T/N)
10
12
Fold change (T/N)
FAM111B
Fold change (T/N)
Fold change (T/N)
Fold change (T/N)
12
1 2 3 4 5 6 7 8 9 10
ZWINT
10
1 2 3 4 5 6 7 8 9 10
Supporting Figure 2 Comparison of mRNA expression levels of genes downregulated
by the KIFC1 inhibitor (kolavenic acid analog) in CRC tissue samples. Fold changes
between CRC tissue (T) and corresponding non-neoplastic mucosa (N) are
demonstrated.
43
ZWINT
KIFC1
10
Negative
Positive
Negative
Weak
10
p = 0.606
CALG
CAHG
p = 0.531
CALG
CAHG
Supporting Figure 3 Summary of immunohistochemical staining of KIFC1 and
ZWINT in conventional colon adenomas. The graph indicates the number of cases with
staining status. CALG, conventional adenoma low grade; CAHG, conventional
adenoma high grade.
...