Download STK31 maintains the undifferentiated state of colon cancer cells

Carcinogenesis vol.33 no.11 pp.2044–2053, 2012
doi:10.1093/carcin/bgs246
Advance Access publication July 25, 2012
STK31 maintains the undifferentiated state of colon cancer cells
Kin Lam Fok1,†, Chin Man Chung1,†, Shao Qiong Yi2,
Xiaohua Jiang1, Xiao Sun1, Hao Chen1,3, Yang Chao
Chen4, Hsiang-Fu Kung5, Qian Tao6, Ruiying Diao1,3,
Henry Chan7, Xiao Hu Zhang1, Yiu Wa Chung1, Zhiming
Cai3 and Hsiao Chang Chan1,*
1
Epithelial Cell Biology Research Centre, The Chinese University of Hong
Kong, Hong Kong SAR, 2Institute of Epidemiology, Academy of Military
Medical Sciences, Beijing, P. R. China, 3Shenzhen Second People’s Hospital,
The First Affiliated Hospital of Shenzhen University, Shenzhen, P. R. China,
4
School of Biomedical Sciences, 5Stanley Ho Centre for Emerging Infectious
Diseases, School of Public Health and Primary Care, 6Department of
Clinical Oncology, The Chinese University of Hong Kong, Hong Kong SAR
and 7Division of Biological Sciences, University of Chicago, IL, USA
*To whom correspondence should be addressed. Tel: +852 26096001;
Fax: +852 26037155;
Email:[email protected]
The expression of serine/threonine kinase (STK) family is frequently altered in human cancers. However, the functions of these
kinases in cancer development remain elusive. Here, we report
that STK31 is robustly and heterogeneously expressed in colon
cancer tissues and plays a critical role in determining the differentiation state of colon cancer cells. Knockdown or overexpression of STK31 induced or inhibited differentiation of colon
cancer cells, respectively. Deletion of the STK domain abolished
the inhibiting effect of STK31. Associated with differentiation,
knockdown of STK31 resulted in significant suppression of tumorigenicity both in vitro and in vivo. Genome microarray analysis
showed that knockdown of STK31 altered the expression profile
of genes that are known to be involved in germ cell and cancer
differentiation. Taken together, these results suggest that STK31
is able to control the differentiation state of colon cancer cells,
which critically depends on its STK domain. The present findings
may shed light on the new therapeutic approach against cancer
by targeting STK31 and cancer differentiation.
Introduction
Cells within a tumor often exhibit variations in biological properties, including the differentiation status, tumorigenecity and
therapeutic resistance. These variables in tumors are referred to as
tumor heterogeneity (1–3). Intratumor heterogeneity has long been
observed in colon cancers (4,5). Recently, using a high throughput
single cell PCR transcription analysis platform, the heterogeneity
in colon cancer has been shown to be a corollary of multi-lineage
differentiation (6), indicating the presence of cellular hierarchy in
colon cancers.
Differentiation of colon cancer cells may give rise to enterocytes in
the adsorptive lineage or enteroendocrine cells and goblet cells in the
secretory lineage (7). TGF-β and BMP pathways are two well-known
cascades that regulate colon differentiation. While TGF-β shows
a strong cytostatic response in intestinal culture in vitro (8), BMP
inhibits intestinal stem cells renewal in vivo (9). These counteracting
cascades share the common SMADs protein family as the intracellular messengers. Although the regulatory machinery in colon cancer differentiation has been postulated to recapitulate the signaling
mechanisms in normal stem cell counterpart (10), the involvement
of reactivated oncogenes in regulating colon-cancer differentiation
through intrinsic mechanism remains elusive.
Abbreviations: STK, serine/threonine kinase; RT-PCR, Reverse transcription
polymerase chain reaction.
†
These authors contributed equally to this work.
Tyrosine kinases and serine/threonine kinases (STKs) are two major
classes of kinases in the human kinome. Although extensive studies
have been focused on the functions and therapeutic potential of tyrosine kinases in cancers (11,12), a recent study revealed that the expression of STKs is frequently altered in human cancers, suggesting that
STKs may play an important role during cancer development. STK31
is a highly conserved member of the STK family and the Tudor Royal
family, containing a STK catalytic domain at the C terminus and a
tudor domain at the N terminus. The expression of STK31 is testis
specific and is restricted to spermatogonia, the stem cell population
in the testis (13). Interestingly, in addition to its specific expression
in the testis, a recent study reported that STK31 was reactivated in
gastrointestinal cancer by promoter hypomethylation, suggesting its
potential role in cancer development (14). Despite all these interesting
findings, the biological function of STK31 in gastrointestinal cancers
has not been investigated. We undertook the present study to investigate the functional role of STK31 and have demonstrated that STK31
plays a critical role in maintaining the undifferentiated state of colon
cancer cells, which critically depends on its STK domain.
Materials and methods
Constructs
Full-length STK31 was cloned into pEGFP-C2 vectors (Clontech, Mountain
View, CA) and verified by sequencing. For domain deletion constructs, 101–
160 a.a. and 809–872 a.a. were deleted in Δtudor and Δkinase, respectively, by
overlapping extension PCR method (15). For RNAi experiment, miR RNA is
were designed using web-based software (Invitrogen, Carlsbad, CA). Design
targeting bacterial LacZ was used as control. Annealed oligos were cloned
into pcDNA6.2 GW/EmGFP-miR vector (Invitrogen, Carlsbad, CA) and verified by sequencing. Primer and oligo sequences are listed in Supplementary
Table 1, available at Carcinogenesis Online.
Gene expressions
RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). Gene
expressions were studied by RT-PCR or real-time PCR using specific primers (Supplementary Table 1, available at Carcinogenesis Online). SYBR
green master mix and 7500 Fast Real-Time PCR Systems were used for realtime-PCR (Applied Biosystems, Carlsbad, CA). Glyceraldehyde 3-phosphate
dehydrogenase was used as internal control.
For microarray analysis, RNA from Caco2 STK31 knockdown and control
miR was labeled with Cy3 and Cy5, with dye swapped in duplicate experiment. Labeled cDNA were hybridized to Agilent human whole genome microarray (Agilent, Santa Clara, CA). Data were analyzed by GeneSpring 10.0
software (Agilent, Santa Clara, CA).
Expressions of candidate genes in germ cells were obtained from
GermSAGE database (16).
Clinical samples
In tissue array experiment, colon adenocarcinoma tissue array was purchased
from US Biomax (US Biomax Inc., Rockville, MD). The tissue array contains
110 colon cancer patients and 10 normal tissues. The 110 patients included 22
well-to-moderately differentiated tumors (Grade 1 or 1–2); 59 moderately differentiated tumors (Grade 2); 22 poorly differentiated tumors (Grade 3) and 7
undefined tumors. The signal intensities were classified into three categories:
(− or 0)––no expression; (+ or 1)––weak expression and (++ or 2)––strong
expression. Since normal tissue sections have signal intensities ≤1, cancer
sections with signal intensities of ≤1 were assigned to low-expression group,
whereas those >1 were assigned to high-expression group. Heterogeneous
expression was defined by the presence of at least 5% of cells with similar
morphology showing differences in signal intensities.
Frozen colon cancer samples were collected from Shenzhen Second
People’s Hospital, the first affiliated hospital of Shenzhen University. Eight
patients were recruited with signed informed consent.
Immunostaining and cell imaging
For immunofluorescent staining in cell lines, Caco2 and SW1116 cells were
grown on coverslips for 48 h and fixed with 4% ice-cooled paraformaldehyde.
In both immunofluorescent and immunhistochemical staining experiments, tissue array or cells were stained with antibody against STK31 (1:100, Abnova,
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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STK31 regulates colon cancer differentiation
Fig. 1. Expression of STK31 in colon cancers. (A) Immunohistochemical staining revealed that STK31 protein was highly expressed in colon cancer patient
samples (representative figures are shown), whereas weak signals were observed in normal tissues. Perinuclear granular staining (dashed circle) was observed.
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Taipei, Taiwan). Slides stained omitting primary antibodies were used as
negative controls. Cell morphologies were analyzed by MetaMorph software.
Three random fields were taken from each stable transfectant. At least six cells
per field were randomly picked and the cell membranes were manually traced.
The number of pixels within the traced area was used for cell-size estimation.
For tumor sections in xenograft experiments, tumor sections were stained
with hematoxylin and eosin. All images were taken by Nikon ELIPSE Imaging
System (Nikon, Melville, NY).
Western blot
Cells were lysed in RIPA buffer (50 mM Tris-Cl pH8.0; 150 mM NaCl; 1%
NP-40; 0.5% deoxycholic acid and 0.1% SDS) with protease inhibitor cocktail.
Proteins were resolved and blotted onto nitrocellulose membrane and probed
with indicated antibodies. Antibodies used in this study were anti-STK31
(1:1000, Abnova, Taipei, Taiwan), anti-cleaved caspase-3, anti-cleaved PARP
(1:500, both from Cell Signaling, Danvers, MA) and anti-β-tubulin, (1:2000,
Santa Cruz Biotech, Santa Cruz, CA).
Cultures and transfection
Caco2 cells were maintained in DMEM supplemented with 10% FBS.
SW1116 cells were maintained in RPMI 1640 supplemented with 10% FBS.
All cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
following manufacturer protocol. In miR RNAi experiment, stably transfected
clones were selected by Blasticidin (Invitrogen, Carlsbad, CA).
In cell-count experiment, 1 × 104 cells per well were seeded into a 24-well
plate. Cells were trypsinized and counted by trypan blue exclusion at indicated
time point.
Flow cytometry
Stable transfectants were seeded into 35-mm dishes at three densities: 1 × 105
per dish (sub-confluent), 6 × 105 (confluent) and 1 × 106 cells (post-confluent).
Cells were grown for 2 days and collected for flow cytometry. Cells were fixed
in chilled 70% ethanol for 30 min on ice. Fixed cell were stained 5 μg/ml propidium iodide (Sigma, St. Louis, MO) and 10 ug/ml RNase A for 2 h at room
temperature. All samples were analyzed by Facscalibur flow cytometer (BD
Biosciences, New Jersey, NJ). Data were analyzed by FCS express V3 software (Denovo software, Los Angeles, CA).
Differentiation assay
In spontaneous differentiation, Caco2 cells were cultured for 14 days with
medium changed every 3 days. In sodium butyrate–induced differentiation,
Caco2 and SW1116 cells were treated with 2 mM sodium butyrate (Sigma,
St. Louis, MO). Cells were collected for molecular studies at 24–48 h
post-treatment.
For alkaline phosphatase activity assay, cells were lysed in lysis buffer (1x
PBS; 5 mM EDTA; 1% Triton X-100 with PMSF and PImix). Protein lysate
was loaded into pNPP substrate solution, incubated for 30 min and terminated
by stopping solution. Optical densities were measured at 405 nm.
Soft agar colony formation assay
A total of 2–4 × 105 cells were mixed with top agar (0.3%) and overlaid onto
bottom agar (0.7%). Colonies were allowed to form for at least 2 weeks followed by staining with 0.005% crystal violet. Number of colonies per plate
was counted. Statistical data were obtained from three biological replicates.
Xenograft assay
Animals were provided by Laboratory Animal Service Centre, CUHK.
The protocols were approved by animal ethics committee, LASEC, CUHK
(09/005/MIS). For Caco2 cells, 6 × 106 cells were injected subcutaneously
into 5–6-week-old female NOD/SCID mice. For SW1116 cells, 1 × 106 cells
were injected subcutaneously into 7–8-week-old female athymic nude mice.
The mice were then killed after 4–6 weeks. Tumor weight, tumor size and the
number of mice that developed solid tumor were determined.
Statistical analysis
Statistical analyses were performed by Prizm 3.0. Results are presented as
mean ± standard error mean (SEM) or standard deviation (SD). Data were
compared by student’s t test, Chi Square (χ2) or one-way ANOVA analysis.
A P value <0.05 was considered statistically significant.
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Results
Reactivation of STK31 in multiple cancers
Previous study has demonstrated the promoter hypomethylation and
reactivation of STK31 in gastrointestinal cancer by PCR and immunohistochemistry (14). To examine the reactivation of STK31 in cancer
development, we studied the expression profile of STK31 in a cohort
of cancers. Our RT-PCR results showed that STK31 mRNA was reactivated in nasopharyngeal and liver cancer cell lines (Supplementary
Figure S1, available at Carcinogenesis Online). The reactivation of
STK31 in a board spectrum of cancers suggests that it may play an
important role in cancer development. To consolidate the reactivation
of full-length STK31 in colon cancer, we audited the protein expression of STK31 by both immunohistochemistry and western blot. We
purchased an antibody raised against human full-length STK31 protein (Abnova) and validated the specificity of the antibody by western
blot in STK31-transfected HEK293 cells. The antibody specifically
recognizes full-length STK31 protein (116 kDa; Supplementary
Figure 2, available at Carcinogenesis Online). Then, we examined
the reactivation of STK31 in a cohort of colon cancer samples by
immunohistochemistry using the validated antibody. Colon tumor tissue array analysis showed enhanced STK31 expression in the cytoplasm of 57% (63 of 110) human colon adenocarcinoma specimens
(Figure 1A; Supplementary Table 2A, available at Carcinogenesis
Online). Weak signals were detected in cancer-adjacent and normal
colonic tissues. Reactivation of STK31 was not always homogeneous within a tumor, with 22 out of 63 STK31-reactivated patients’
samples showing varied signal intensities in at least 5% of the cancer cells in the samples. The expression of STK31 appeared to be
less heterogeneous in moderately to poorly differentiated cancers
(31% heterogeneous expression) when compared with well-differentiated cancers (50% heterogeneous expression; P = 0.28); however, the difference was not statistically significant (Supplementary
Table 2B, available at Carcinogenesis Online). While the reactivation of STK31 was not correlated to gender and histological grade
(P = 0.25 and 0.23, respectively), patients with STK31 reactivation
were slightly younger (P = 0.16; Supplementary Table 2B, available at Carcinogenesis Online), which is consistent with the previous
report (14). Interestingly, perinuclear granular signals were observed
in some of the tumor samples (Figure 1A red circle), which is reminiscent of the localization of its mouse homolog—Stk31—in the RNA
processing body of the germ cells (17). To further validate the reactivation of STK31 in a quantitative way, we examined the expression
of STK31 by real time-PCR. The results showed that the expression of
STK31 transcripts were significantly increased in 5 out of 8 patients
(P < 0.05) compared with normal tissue (n = 8; Figure 1B), confirming the reactivation of STK31 in colon cancer.
We next examined the expression and localization of endogenous
STK31 in two colon cancer cell lines—Caco2 and SW1116—by
immunofluorescent staining. Consistent with the IHC results, STK31
was localized in the cytoplasm (Figure 1C). Of note, expression of
STK31 was heterogeneous in these two cancer cell lines. Particularly,
in Caco2 colonies, high STK31 expression levels were found at the
edge of colonies, which became decreased or undetectable in the
central part of the colonies, where cells were compacted and were in
contact with each other. Given that Caco2 cells are prone to undergo
spontaneous post-confluence differentiation (18), the marked decrease
of STK31 expression in the confluent cell population suggests that the
expression of STK31 is downregulated in a differentiated state. To
further demonstrate the correlation between the expression of STK31
and the differentiation of colon cancer cells, Caco2 cells were treated
Magnification: ×200 (left panel) and corresponding enlarged images were shown (right panel). Scale bar: 50 µm. (B) Quantitative PCR analysis of STK31 in
colon cancer patients. (C) Immunofluorescent staining of STK31 in Caco2 and SW1116 colon cancer cells. STK31 was heterogeneously expressed within a
cell line. In Caco2 cells, expression of STK31 was mainly detected on the edge of a colony and decreased in the central population (arrow). In SW1116 cells, a
small population of flattened cells (asterisk) demonstrated weaker STK31 expression. Scale bar: 50 µm. (D, E) Alkaline phosphatase activities (D) and protein
expression of STK31 (E) in Caco2 cells treated with sodium butyrate. Data are presented as mean ± SEM; t test, *P < 0.05, **P < 0.01.
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Fig. 2. Knockdown of STK31 alters cell morphology and suppresses cell proliferation and cell cycle progression in both Caco2 and SW1116 cells. STK31 RNAi
construct was stably transfected into Caco2 and SW1116 cells. (A) Western blot of STK31 showed that the RNAi design could knock down STK31 in both cell lines.
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with sodium butyrate, a metabolite that is known to induce differentiation of colon cancer cells. After sodium butyrate treatment, the
activity of the brush border enzyme—alkaline phosphatase—was
significantly increased (Figure 1D) while the protein expression of
STK31 was markedly downregulated (Figure 1E). This result suggested a negative regulation of STK31 on differentiation of colon
cancer cells.
Knockdown of STK31 alters cell morphology and cell cycle
progression
Differentiation of colon adenocarcinoma cells is characterized by cell
flattening (19) and growth retardation (20,21). To test whether STK31 is
involved in regulating cell differentiation, we designed and constructed
an RNAi for stable knockdown of STK31 in Caco2 and SW1116
cells (Figure 2A). Growth and morphology of the gene-manipulated
cells were monitored over time. While knockdown of STK31 did not
alter morphological or growth properties in subconfluent cultures
(Supplementary Figure 3, available at Carcinogenesis Online), cells
with knocked-down STK31 became significantly more flattened and
spread out on culture dish upon cell confluence as indicated by the
average area taken up by a cell (P < 0.01; Figure 2B). The flattened
morphology could not be attributed to the increase in cell size as
indicated by flow cytometric analysis (Supplementary Figure 4,
available at Carcinogenesis Online). More importantly, knockdown of
STK31 resulted in a significant decrease in cell proliferation in both
cell lines upon confluence (P < 0.05; Figure 2C). Further analysis
on cell cycle progression in Caco2 cells revealed that knockdown of
STK31 resulted in G1 arrest in both confluence and post-confluence
(P < 0.05; Figure 2D). These results indicate that knockdown of
STK31 has the potential to enhance differentiation in both colon
adenocarcinoma cell lines.
Knockdown of STK31 enhances differentiation of colon cancer cells
When grown in appropriate culture conditions or treated with inducers, colon cancer cells from primary tumors can differentiate into two
lineage-restricted cell types after in vitro expansion—the enterocytes
in the absorptive lineage and the goblet cells in the secretive lineage
(7,22). Caco2 cells are capable to undergo enterocytic differentiation
that is marked by the formation of dome and the expression of villin
(an enterocyte marker), whereas SW1116 cells are capable to undergo
goblet-like differentiation that is identified by expression of mucin (a
goblet cell marker; 7,23,24). Since transition from undifferentiated
to differentiated states in these two cell lines seems to recapitulate
the growth arrest and commitment of lineage-specific differentiation
of colonic cancer cells, we further investigated the potential roles of
STK31 in regulating differentiation of the colon carcinoma cell phenotype in these two cell lines.
In spontaneous differentiation of Caco2 cells (25), knockdown of
STK31 increased both dome (P < 0.05) and pleomorphic glandular
formations (P < 0.001; Figure 3A), which are indicative of mature phenotypes of colon cancer cells (22). In keeping with the morphological
changes, expression of villin was ~2–3-fold higher in STK31 knockdown cells compared with that in control cells in both spontaneous
and sodium butyrate–induced differentiations (P < 0.05; Figure 3B).
Besides, in sodium butyrate–induced differentiation of SW1116 cells,
knockdown of STK31 resulted in significant increase in the number
of cells with vacuolated cytoplasm (P < 0.001; Figure 3C), a morphological characteristic of goblet-like differentiation (7,26). Although
differentiation marked by vacuolated cytoplasm had been associated with senescence, it should not have been the case here because
←
no massive cell death or senescence was observed (Supplementary
Figure 5, available at Carcinogenesis Online). In addition, expression
of mucin was significantly higher (~2–3-fold) in STK31 knockdown
cells compared with that in control cells (P < 0.01; Figure 3D). These
results suggest that STK31 may be required for maintaining the undifferentiated state of the two colon cancer cell lines.
STK domain of STK31 is required for maintaining the undifferentiated
state
To confirm the role of STK31 in maintaining the undifferentiated
state of colon cancer cells, we examined whether the overexpression
of STK31 would inhibit differentiation. To further identify the functional domains on STK31 responsible for regulating differentiation,
we constructed window-deletion mutants in two conserved domain,
the N-terminal tudor domain (ΔTudor) and the C-eterminal kinase
domain (ΔKinase), of STK31 (Figure 4A). Since the transfection
efficiency of Caco2 cells is low, we used SW1116 cells for transient
transfection experiments. To induce differentiation, sodium butyrate
was added to the transfectants 24 h post-transfection. Overexpression
of STK31 and the two mutants was confirmed by western blot
(Figure 4B). Differentiation status of transient transfectants was determined by the expression of goblet cell differentiation marker—mucin.
As expected, the overexpression of STK31 significantly decreased the
expression of mucin in both untreated and butyrate-treated conditions (Figure 4C, P < 0.001 and P < 0.01, respectively), indicating
that the STK31-overexpressed groups are less differentiated in both
conditions. This result confirmed the role of STK31 in maintaining
the undifferentiation state of colon cancer cells. In domain deletion
experiment, the expression of mucin in Δtudor mutant-transfected
cells was similar to that of full-length STK31 transfectant, suggesting
that tudor domain was dispensable in regulating the differentiation
of cancer cells (Figure 4C). Interestingly, deleting the STK domain
partially reversed the inhibition of differentiation in untreated condition while completely abolishing the effect of STK31 overexpression
on butyrate-induced differentiation (Figure 4C). These results suggest
that the STK domain of STK31 is required for regulating the differentiation of colon cancer.
Knockdown of STK31 suppresses tumorigenicity in vitro and in vivo
The observed inhibition in cell proliferation and enhanced differentiation in STK31 knockdown colon cancer cells prompted us to assess
the tumorigenicity of STK31 knockdown colon cancer cells in vitro
and in vivo by soft agar colony formation assay and mice xenograft
assay, respectively. As shown in Figure 5A, the knockdown of STK31
significantly decreased the number of colonies formed in soft agar
in both CaCo2 and SW1116 cells (P < 0.05). In SW1116 xenografts, although all mice developed solid tumors, the size of tumors
in STK31 knockdown group was significantly smaller than those in
the control group (P < 0.05; Figure 5B). Moreover, histochemical
staining showed that STK31 knockdown tumors displayed a more
differentiated phenotype characterized by the formation of a glandular structure (Figure 5C). Similar results were obtained in Caco2
xenograft experiment where knockdown of STK31 decreased the
number of mice developing solid tumors (Supplementary Figure 6A,
available at Carcinogenesis Online). Furthermore, while sections of
tumor formed by control miR Caco2 cells demonstrated typical welldifferentiated adenocarcinoma morphology, tumor formed by STK31
knockdown Caco2 cells showed remarkable karyorhexis and karyolysis, indicating the occurrence of necrosis (Supplementary Figure 6B,
available at Carcinogenesis Online). Together, these results indicate
(B) Live cell images of Caco2 and SW1116 stable transfectants showed that the knockdown of STK31 (STK31 kd) resulted in cell-flattened morphology in both
cell lines (inset) at post-confluence stage, compared with control (Ctl) RNAi. Magnification: ×100. Quantitative analysis of occupied area by one cell in 2D
cultures. The area occupied by STK31 knockdown transfectant in 2D cultures was significantly larger than that in control RNAi (P < 0.001). (C) Cell counting of
both control (ctl RNAi) and STK31 knockdown cells (STK31 kd) from Caco2 and SW1116 cultures showed significant decrease in cell proliferation in STK31
knockdown cells after the culture reached confluence (P < 0.05). Data are presented as mean ± SEM; Student t test. (D) Knockdown of STK31 in Caco2 led to a
G1/S arrest in confluent and post-confluent culture. Data are presented as mean ± SD; t test, *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 3. Knockdown of STK31 enhances differentiation of Caco2 and SW1116 cells. 6 × 104 stable transfected Caco2 and 4 × 104 stable transfected SW1116
cells/well of a 24-well plate cells/well of a 24-well plate were cultured in a serum-containing medium for 14 days for spontaneous differentiation or treated
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Fig. 4. Effect of domain deletion and overexpression of STK31 on SW1116 differentiation. (A) Schematic diagram on the window deletion of STK31. (B)
Western blot showing the overexpression of full-length and domain-deleted STK31 in SW1116 cells. The size of Δtudor and Δkinase are slightly smaller than that
of the full-length protein. (C) Real time-PCR results showing the expression of goblet cell marker, mucin, was downregulated in STK31-overexpressed SW1116
cells under either untreated or sodium butyrate–induced differentiation condition. Δtudor-expressed cells showed similar mucin expression compared with that
of full-length STK31 overexpressed cells. Deleting the STK domain completely abolished the down-regulation of mucin in sodium butyrate–induced condition.
Data are presented as mean ± SEM; One-way analysis of variance, *P < 0.05, **P < 0.01, ***P < 0.001.
that the knockdown of STK31 decreases tumorigenicity both in vitro
and in vivo by promoting differentiation of colon cancer cells.
Differential gene expression in STK31 knockdown-mediated colon
cancer differentiation
We next asked how STK31 could regulate the differentiation status of
colon cancer. To answer this question, we compared the transcriptome
of STK31 knockdown and control Caco2 cells using whole genome
microarrays, and identified 95 genes that were significantly altered
(≥2-fold). Interestingly, among the 95 genes regulated by STK31, 10
genes (KIT, NRG1, HDGFRP3, SMAD1, AR, GJA1, CCND2, TREFR1,
FABP5 and SCIN) have been implicated both in tumorigenesis and in
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spermatogenesis (Supplementary Table 3, available at Carcinogenesis
Online). To validate that genes identified by microarray study are
bona fide targets of STK31, we performed real time-PCR to assess the
expression levels of genes that (i) their function in tumorigenesis has
been reported and with a 10-fold difference in microarray study or (ii)
are known to implicate in both spermatogenesis and tumorigenesis.
Our results showed that AR, FGF12, GJA1, HDGFRP3, LUM, NRG1,
and SMAD1 were significantly downregulated in STK31 knockdown
cells from both Caco2 and SW1116 cell lines (Figure 6A and 6B).
Downregulation of KIT was only observed in STK31 knockdown
Caco2 cells, but not in SW1116 cells probably because SW1116 do
not express this receptor. Differential expression of the other two
with 2 mM sodium butyrate (NaBT) for 48 h. (A) Knockdown of STK31 enhanced enterocytic differentiation in Caco2 cells as reflected by significant increase in
number of dome (dash circles) and glandular crypt formation (arrows). Magnification: ×100. Quantification of data is shown on the right panel. (B) Real time-PCR
showed that the expression of an enterocyte marker, villin, was up-regulated in STK31 knockdown Caco2 cells that underwent spontaneous (left) and sodium
butyrate–induced differentiation (right) compared with control cells. Data are presented as mean ± SEM; t test, **P < 0.01, ***P < 0.001. (C) Knockdown of
STK31 enhanced goblet-like differentiation in SW1116 cells as indicated by significant increase in the number of cells showing vacuole formation in cytoplasm
(arrows). Magnification: ×100 (left panel) and ×200 (right panel). Quantification of data is shown on the right panel. (D) Real time-PCR showed that expression of
goblet cell marker, mucin, was up-regulated in STK31 knockdown SW1116 cells that underwent sodium butyrate–induced differentiation. Data are presented as
mean ± SEM; t test, **P < 0.01, ***P < 0.001.
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Fig. 5. Knockdown of STK31 decreases tumorigenecity with enhanced differenitation. (A) Knockdown of STK31 in both Caco2 and SW1116 cell lines
decreases the number of colonies formed in soft agar colony formation assay. Quantification of data is shown below. (B) 1 × 106 STK31 knockdown SW1116
cells were subcutaneously grafted into athymic nude mice. Mice were killed 2 weeks later and the tumors were collected and dissected for histochemical staining.
Knockdown of STK31 in SW1116 cells decreased the size of tumor formed (n = 8). (C) Tumor xenograft formed by STK31 knockdown SW1116 cells showed
a higher differentiation status compared with that formed by control cells as indicated by the presence of glandular cavities (arrow and inset; scale bar: 100 µm).
Data are presented as mean ± SEM; t test, *P < 0.05, ***P < 0.001.
genes—ARL11 and PTPN13—was also observed in Caco2 cells only
(Figure 6A and 6B).
Discussion
The present study has demonstrated that STK31 is highly expressed
in multiple cancers, including colon cancer. Suppression of STK31
induces differentiation and inhibits tumorigenecity of colon cancer
cells, both in vitro and in vivo. In general, tumors are heterogeneous in
terms of their surface antigen expression, cell morphology (differentiation stages), proliferation, metastatic capacity, tumorigenecity and
therapeutic resistance (1,2), indicating a cellular hierarchy consisting of cancer cells at various stages of abnormal differentiation. This
cellular hierarchy is thought to be tightly regulated by homeostasis
between proliferation and differentiation (27); however, the detailed
regulatory mechanism remains unclear. Here, we have demonstrated
that the reactivation of STK31 is crucial in maintaining the undifferentiated state of colon cancer cells. The heterogeneous expression pattern of STK31 in both cancer cell lines and primary tumors,
together with the enhanced differentiation upon STK31 knockdown,
strongly suggests its possible role in maintaining the primitive state
of its expressing cells and thus the cellular hierarchy within the colon
cancer cell lines or primary tumors. Moreover, the suppressed cancer
growth associated with differentiation in vivo further lends compelling support for the anti-differentiation role of STK31 in colon cancer.
STK31 contains two highly conserved domains: a tudor domain
and a STK domain. Of interest, we found that the STK domain is
required for regulating the differentiation of colon cancer cells while
the tudor domain is dispensable for the process. It has been reported
that the expression of STKs is frequently altered in human cancers (28). Importantly, the majority of the STKs are reactivated or
up-regulated in cancers, suggesting that gaining the activities of STK
may favor tumor development. In the present study, we have demonstrated for the first time that the STK domain of STK31 is required
for maintaining the undifferentiation state of colon cancer, providing
strong support to the importance of STK in regulating tumor development. Since a variety of inhibitors of STKs is available (29), they may
be used for cancer therapy in targeting reactivated or up-regulated
STKs, including STK31.
The restricted expression in normal testis and aberrant expression
in many tumors indicates that STK31 can be classified as cancer/testis
gene. It has been suggested that spermatogenesis and tumorigenesis
share significant similarities in biological and biochemical processes
(30), which may be attributable to the reactivation of cancer/testis
genes. Previous reports and our preliminary data revealed that STK31
expression was confined to spermatogonia in testis (13), indicating its
pivotal role in germ cell differentiation. Therefore, we speculate that
the regulation in colon cancer differentiation may resemble that in
germ cell differentiation. In support of this notion, our gene expression profiling studies revealed that ~40% (39/95) of the STK31 target
genes identified in colon cancer cells were expressed in germ cells. It
is particularly noteworthy that several important autocrine/paracrine
systems known to be involved in spermatogenesis (including KIT/
SCF, BMPs/SMADs, NRG1/ErbB and possibly hepatoma-derived
growth factor HDGF/HDGFRP3) are regulated by STK31 in colon
cancer cells. Moreover, most of the STK31 targets (KIT, SMAD,
NRG1) in colon cancer cells are specifically expressed in spermatogonia in testis, in which STK31 is normally expressed, and has been
implicated in the germ cell differentiation. During spermatogenesis,
the receptor tyrosine kinase, KIT, and its ligand, stem cell factor
(SCF), are crucial for spermatogonial proliferation and differentiation
(31). Knockout of KIT has been shown to abolish spermatogonial
2051
K.L.Fok et al.
Fig. 6. Candidate genes regulated by STK31 in Caco2 and SW1116 cells. (A, B) Differentially expressed genes in STK31 knockdown Caco2 cells (A) were
identified by whole genome microarray and confirmed by real time-PCR. (B) Differential expressions of candidate genes were confirmed by real time-PCR in
SW1116 cells. Data are presented as mean ± SEM; t test, *P < 0.05, **P < 0.01, ***P < 0.001.
stem-cell differentiation, which results in sterility (32). Signaling by
KIT also plays an important role in cellular transformation and differentiation through multiple signaling cascades, including STAT3,
PI3K, PLC and MAPK (33–36). Of particular relevance to this study,
human colorectal tumors express c-kit and activation of c-kit protected human colon adenocarcinoma cells against apoptosis enhanced
their invasive potential (37). In our study, the downregulation of KIT
by STK31 knockdown suggests that repression of KIT may be functionally important in STK31-mediated regulation of differentiation
and tumorigenicity in colon cancer. Apart from KIT, BMP signaling plays critical roles both in colon cancer progression (38) and in
spermatogonial differentiation (39). Particularly, SMAD1-mediated
signaling cascades have been implicated in anchorage-independent
growth (40) and tumorigenicity (41). Consistent with these findings,
we have found that SMAD1 was downregulated by STK31 knockdown in the two colon cancer cell lines (Figure 6), indicating that
SMAD1 is another possible target of STK31 contributing to its suppressive effects on tumorigenicity in vitro and in vivo.
In conclusion, our study suggests that reactivation of STK31 in
colon cancer maintains the undifferentiated state of colon cancer cells.
Acquisition of this anti-differentiation ability could be one of the driving
forces of tumorigenesis in colon cancer development. The current findings are of important clinical implications. Traditional cancer therapy has
limitations with chemoresistance and cancer relapse, which are largely
responsible for the treatment failure (10). With better understating on the
molecular mechanisms underlying cellular heterogeneity and hierarchy
in tumors, new therapeutic approach targeting undifferentiated tumorigenic tumors cells has evolved. The so-called differentiation therapy
2052
attenuates the tumorigenicity of cells with high differentiation potential
through enforced commitment into defined lineages (42,43). Given its
demonstrated role in regulating differentiation, together with its expression in a broad spectrum of cancers and specific expression in normal
testis, STK31 represents a potent and promising target for the development of differentiation-directed therapy in colon cancer treatment.
Supplementary material
Supplementary Tables 1–3 and Figures 1–6 can be found at http://
carcin.oxfordjournals.org/
Funding
This work was supported by National 973 program (2012CB944903),
the Li Ka Shing Institute of Health Sciences and the Focused
Investment Scheme of the Chinese University of Hong Kong.
Acknowledgements
The authors thank Prof XY Guan (Department of Clinical Oncology, The
University of Hong Kong) and Prof KW Lo (Department of Anatomical and
Cellular Pathology, The Chinese University of Hong Kong) for providing the
cell lines cDNA for expression profiling.
Conflict of Interest Statement: None declared.
STK31 regulates colon cancer differentiation
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Received April 11, 2012; revised June 28, 2012; accepted July 17, 2012
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