Review

The Identifications and Clinical Implications of

Cancer Stem Cells in Colorectal Cancer
S.M. Riajul Wahab,1 Farhadul Islam,1 Vinod Gopalan,1,2 Alfred King-yin Lam1
Abstract
Cancer stem cells (CSCs) are cancer cells that are responsible for initiation, progression, metastasis, and recurrence in
cancer. The aim of this review was to analyze the markers for identifying of CSCs in colorectal carcinoma, as well as
the prognostic and therapeutic implications of these markers in the cancer. CSCs are insensitive to the current drug
regimens. In colorectal carcinoma, markers, including Nanog, Oct-4, SOX-2, Lgr-5, CD133, CD24, CD29, ALDH1,
EpCAM, CD44, CD166, and CD26, are commonly used for the identification and isolation of CSCs. In addition, ALDH1,
CD24, CD44, CD133, CD166, EpCAM, Lgr-5, Nanog, and SOX-2 could have clinical roles in predicting pathological
stages, cancer recurrence, therapy resistance, and patients’ survival in patients with colorectal carcinoma. In light of
the current knowledge of CSCs in colorectal carcinoma, novel potential therapeutic strategies, such as development
of monoclonal antibodies or immunotoxins and targeting various cell surface molecules in colorectal CSCs and/or
components of signaling pathways, have been developed. This could open new opportunities for the better man-
agement of patients with colorectal carcinoma.

Clinical Colorectal Cancer, Vol. 16, No. 2, 93-102 ª 2017 Elsevier Inc. All rights reserved.
Keywords: Cancer, Carcinoma, Markers, Stem cell, Therapy

Introduction morphologically diverse cells, including therapy-resistant and met-

Colorectal cancer (CRC) is the third most commonly diagnosed astatic cell populations.3
cancer in men and the second in women. In 2012, there were an CSCs have been implicated in colorectal carcinogenesis for a long
estimated 1.4 million incidences and 693,900 deaths throughout time, although their existence has been only recently demonstrated
the world.1 Failure of treatment of patients with CRC could be experimentally.9,10 In view of the importance of CSCs in CRC, we
attributed to the escaped residual microscopic carcinoma after sur- aimed to review the markers for identifying of CSCs in CRC as well
gery, which later initiates the metastatic process.2 In principle, these as the prognostic and therapeutic implications of these markers
residual cancer cells are eliminated by postoperative chemotherapy in CRC.
and/or radiotherapy. However, the presence of therapy-resistant
cancer cells limits the success of these treatments.3,4 Genetic, Identification of CSCs
epigenetic, and functional heterogeneity of cancer cells supports the By definition, CSCs are the cells that have the capacity to drive
existence of these therapy-resistant cancer cells in patients with carcinogenesis through long-term production and self-renewal of
CRC.5-7 These small fractions of cells within the cancers are called differentiated, nontumorigenic progenies.11 It was also reported that
cancer stem cells (CSCs). These CSCs are capable of initiating, chemoradiotherapy-resistant CSCs have greater potential of tumor
maintaining, and developing cancer growth.3,8 Also, CSCs have self- initiation and stimulated the regrowth of cancer after a therapeutic
renewal capacity and are responsible for developing functionally and treatment.12-15 The existence and the identity of CSCs have been
reported for the first time in hematopoietic cancers.16 Thereafter,
CSCs from many solid cancers, such as those arising from breast,
1
Cancer Molecular Pathology, School of Medicine brain, prostate, head, and neck, were also identified.12,13,17
2
School of Medical Science, Menzies Health Institute Queensland, Griffith University,
Gold Coast, Queensland, Australia
The current gold standard for defining CSC “stemness” is to
show their ability to transfer disease into immunodeficient mice at a
Submitted: Jul 31, 2016; Revised: Nov 16, 2016; Accepted: Jan 13, 2017; Epub:
Jan 25, 2017
limited dilution.14,15 This type of xenograft assay involves
fluorescence-activated cell sorting of a single cancer cell that has the
Address for correspondence: Professor Alfred King-yin Lam, MBBS, MD, PhD,
FRCPA, Head of Pathology, Griffith Medical School, Gold Coast Campus, Gold putative CSC properties and demonstrates its ability to develop a
Coast, Queensland 4222, Australia new cancer similar to the original cancer.14,15,18 The limitation of
E-mail contact: a.lam@griffith.edu.au
this method is partly related to the difficulties discriminating

1533-0028/$ - see frontmatter ª 2017 Elsevier Inc. All rights reserved.

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 Cancer Stem Cells in Colorectal Cancer
between CSC and non-CSC populations of cancer cells. Also, the some cells within the mouse colonic adenoma (5%-10%) were Lgr-
difference between the microenvironment of the original cancer and 5þ cells. These cells were responsible for self-renewal and produc-
the transplanted recipient may have an impact on the function of tion of differentiated Lgr-5 colonic adenoma cells. It was reported
CSCs.19 Thus, the identification and isolation of a CSC is still a that patients with CRC expressing high Lgr-5 had 10-fold higher
matter of debate due to lack of unique methods for isolation and risk for cancer relapse than patients with low expression of Lgr-5.39
identification as well as their complex biology.9,10 In addition, it has been demonstrated that Lgr-5þ cells derived
from patients with CRC have the potential of CSC as they showed
Identification of CSCs in CRC high number of spheroid formation in culture conditions.41
Genes such as Nanog, Oct-4, and SOX-2 are responsible for the Therefore, Lgr-5 has the potential to be used as a surrogate
pluripotency of cells and are commonly considered to be the sur- marker for the identification of CSCs in CRC.
rogate markers for CSCs (Table 1).20,21 Cluster of differentiation 24 (CD 24), also called heat stable
Nanog, a homeobox protein encoded by Nanog, is a transcription antigen 24 (HAS) or signal transducer 24, is a glycoprotein and
factor and regulates the stem cell properties, especially the self- expressed at the cell surface of lymphocytes.42 Rowehl and col-
renewal pluripotency of cells.22 Matsuoka and colleagues23 showed leagues43 reported the establishments of CRC’s CSCs using in vitro
that nanog was positive in 28 (10%) of 290 gastric cancer tissues. In and in vivo mouse models from liver metastasis of patients with
CRC, Meng and colleagues24 highlighted the importance of Nanog colon cancer. This study also demonstrated that CD24þ cells were
in the maintenance of cell proliferation, invasion, and motility of highly tumorigenic and clonogenic with increased stemness and
CRC cells as well as its contribution to the epithelial mesenchymal pluripotency and exhibited resistance to therapy.43 Sahlberg and
transition (EMA) in the development of CRC. colleagues44 reported that colon cancer cells expressing CD24,
Oct 4 (a member of the POU family) contributes to the self- CD133, and CD44 act as CSCs and was associated with radiation
renewal ability and inhibits the genes responsible for differentiation resistance in colon cancer cells. Thus, CD24 can be used as a pu-
as well as to enable the self-renewal ability of stem cells.25,26 Padín- tative marker for CSC isolation and identification in CRCs.
Iruegas and colleagues27 demonstrated that Oct4 mRNA was present CD29, also called integrin beta-1 protein, is encoded by the
in the peripheral blood of patients with metastatic CRC. ITGB1 gene. It plays a key role in cell adhesion and various cellular
Sex-determining region Y (SRY)-box 2 (SOX-2) is a stem cell processes like embryogenesis, hemostasis, tissue repair, immune
marker and plays crucial roles in the maintenance of cell pluripo- response, and cancer metastases.45 CD29 is reported to be a surface
tency and self-renewal.28-30 In addition, it has been reported that marker for the highly proliferative site of human colonic crypt, and
SOX-2 plays an important role in the maintenance of self-renewal of thereby CD29-positive cells could be used as a marker for stem cell
CSCs.31 Knockdown of SOX-2 and Oct4 reduced the tumor size in type in human colon.46 In addition, high expressions of CD29 were
oral cancer in immunodeficient mice.32 Furthermore, SOX-2 was noted in human colon CSCs and these cells acted as tumor initiator/
found positive in 159 (55%) of 290 gastric cancers.23 In CRC, CSCs in mouse colonic carcinoma.47 Another study has identified
SOX-2 has been used to identify the CSCs in many studies.33-35 that colon CSCs with phenotypic fractions of CD29þ/CD133þcells
O’Brien and his group15 noted that CD133-positive human exhibited distinct proliferation, differentiation, and self-renewal
cancer cells were able to produce cancer of similar morphology to properties.48 These studies suggest that CD29 can be used as a
the original one in immunodeficient mice, whereas the CD133- surface marker in identifying CSCs in colon cancers.
negative cells were unable to initiate cancer growth. CD133 has Aldehyde dehydrogenase isoform 1 (ALDH1) is an isoform of
been used to study 501 CRC on tissue microarrays in CRC.34 aldehyde dehydrogenase enzyme and catalyses the conversion of
Leucine-rich repeat-containing G-proteinecoupled receptor 5 aldehyde to carboxylic acid.49 This enzyme is commonly used as a
(Lgr-5)epositive cells (Lgr-5þ) have the characteristic features of surrogate marker for the identification of non-CSCs as well as CSCs
CSCs in CRC.36-40 Schepers and colleagues38 demonstrated that in different cancers, including breast cancer, pancreatic cancer,

Table 1 Biomarkers of Colorectal Cancer Stem Cells

Protein Markers Gene Assay Method References

Nanog, Oct-4, Nanog, POU5F1, SOX-2 Therapy-resistant assay; quantitative reverse transcriptase polymerase 20-24,33
SOX-2 chain reaction
CD133 PROM1 Chemoresistance assay; colony formation assay 9,15,34,98-100
Lgr-5 LGR5 Tumorigenicity assay; experimental metastasis assay 40,47
CD24 CD24 Colony formation assay; invasion assay; differentiation assay; survival assay 47,84,85
CD29 ITGB1 Colony formation assay 47
ALDH-1 ALDH1A1 Xenotransplantation in immunodeficient mice 51,72
EpCAM EPCAM Immunohistochemistry; Western blot assay 58,98
CD44 CD44 Xenotransplantation in immunodeficient mice; colony formation assay 47,58,88,136
CD166 ALCAM Tumor growth in immunodeficient mice following xenograft; colony 47
formation assay
CD26 DPP4 Tumor formation and metastasis following xenotransplantation 69

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 S.M. Riajul Wahab et al
prostatic cancer, lung cancer, leukemia, multiple myeloma, mela- CD26 cells. Furthermore, they found that transient knockdown of
noma, and liver cancer.11,17 Studies have noted that ALDH1 is a CD26 in CD26þ cells decreased the migratory and invasive capacity
potential CSC marker in CRC.50,51 Increased ALDH1 expressions of CD26þ CSCs.
in colon cancer tissue samples were associated with poor differen-
tiation (high grade) and presence of metastasis.52,53 Prognostic Value of CSC Markers in
Epithelial cell adhesion molecule (EpCAM) is a transmembrane CRC
glycoprotein, mediates homotypic cell-cell adhesion in the epithelia, CSCs can regulate cancer invasion, distant metastases, therapy
and regulates cell proliferation, differentiation, migration, and cell resistance in CRC, as well as contribute to the cancer recurrence of
signaling.54,55 This cell surface marker has the potential to be used patients with CRC.70 Taken together, the markers for CSCs could
as a diagnostic marker for detecting carcinomas.54 Roy and co- potentially have important implications in the prognosis of patients
workers56 isolated colonic CSCs using EpCAM, CD133, and with CRCs (Table 2).
CD44 cell surface markers in the xenograft CSC mouse model.
CD44 is a cell surface glycoprotein encoded by CD44 gene and ALDH1
regulates cell-cell interactions, cell adhesion, and migration.57 CSCs High expression of DNA repair mechanism, aldehyde dehydro-
from different cancers, including colon cancer, breast cancer, genase isoform 1 (ALDH1) and other molecular pumps, such as
pancreatic cancer, head and neck cancer, hepatocellular carcinoma, ATP-binding cassette transporter (ABC-transporter) in CSCs,
and nonesmall-cell lung cancers have been identified and isolated contribute to overcome the effect of chemoradiotherapy in
using CD44.58 It was reported that CD44þ CRC cells exhibited CRC.66,70 Increased ALDH1 expressions in colon cancer tissue
higher in vitro clonogenic properties and showed higher in vivo samples were associated with advanced clinical stage.52 Lugli and
tumorigenicity when compared with that of CD44 cells.59 coworkers53 demonstrated that overexpression of ALDH1 protein
Furthermore, CD44þ CRC cells displayed the phenotypic and in primary colorectal adenocarcinoma tissues (n ¼ 1420) via im-
morphological characteristics of cancer following serial trans- munostaining was associated with high pathological grade and poor
plantation into immunodeficient animals.59 These CD44þ cells survival of the patients. Similarly, Fitzgerald and colleagues71 re-
maintained their stemness by activation of tyrosin kinase receptor c- ported that high ALDH1 protein expression, detected by immu-
Met in colon cancer.59 Dalerba and colleagues58 demonstrated that nohistochemistry, in stage IV colorectal adenocarcinoma tissues
triple-positive surface phenotype (EpCAMhigh/CD44þ/CD166þ) (n ¼ 30) was correlated with poor survival of the patients. Recently,
could be used as a precise method for identification of colonic Deng and coworkers72 reported that patients with rectal cancer (n ¼
CSCs. 64) receiving preoperative radiochemotherapy showed high expres-
Wang and colleagues60 noted that CD44þ colon cancer cells sion levels of different CSC markers, including ALDH1 by im-
displayed more aggressive proliferation, higher colony formation, munostaining. They noted that high ALDH1 expression in patients
less sensitive to apoptosis signals, and more resistance to therapy with post-neoadjuvant therapy correlated with cancer relapse,
when compared with that of CD44 cells. However, studies noted distant metastasis, and poor prognosis in rectal cancer.72 Also,
a controversial role of CD44 in the pathogenesis of CRC.61,62 Goosssens-Beumer and coworkers73 studied the expression of
Dallas and coworkers61 noted that downregulation of CD44 ALDH1 in a large cohort of patients (n ¼ 309) with CRC by
increased migration and metastasis of colon cancer cells. Also, loss of immunohistochemistry and was significantly correlated with poor
CD44 was noted to be correlated with aggressiveness of colon clinical outcome of the patients. Furthermore, Kahlert and col-
carcinomas as reported by Ylagan and colleagues.62 Thus, further leagues74 noted that ALDH1 nuclear expression was associated with
studies are needed to confirm the role of CD44 in the maintaining shortened overall survival of patients with CRC. Therefore, these
of stemness of colon CSCs. studies indicated that ALDH1 acts as a strong prognostic marker in
CD166, also called activated leukocyte adhesion molecule patients with CRC.
(ALCAM), is a transmembrane glycoprotein in the immunoglob-
ulin superfamily. It is encoded by ALCAM and characterized by the CD24
5 extracellular immunoglobulinlike domains.63 CD166 is expressed CD24 is a glycosylphosphatidylinositol-anchored membrane
high in colon cancer, lung cancer, breast cancer, melanoma, and protein and acts as an adhesive molecule on the activated endothelial
prostate cancer cells.64 Several studies demonstrated the identifica- cells and platelets.75-78 Studies demonstrated that CD24 is a po-
tion of colorectal CSCs using CD166 as a cell surface marker.65,66 tential prognostic marker in various cancers, such as ovarian cancer,
For instance, Margaritescu and colleagues65 reported the identifi- nonesmall-cell lung cancer, prostatic cancer, gastric adenocarci-
cation of colon carcinoma stem cells using CD133/CD166/Ki-67 noma, and breast cancer.79-83
triple-positive phenotype in immunofluorescence techniques. Weichert and colleagues84 showed strong cytoplasmic CD24
These results were in consensus with the earlier findings of Dalerba expression in CRCs and the expression of CD24 was associated with
and coworkers66 in which colonic CSCs were isolated using shortened patient survival in patients with CRCs. Also, Choi and
CD166. coworkers85 showed that CD24 expression was related to the his-
CD26 is a cell surface glycoprotein that is expressed in a variety of tological grade and size of the CRC.
cell types, including endothelial cells, epithelial cells, and T lym- Seo and colleagues86 examined 174 stage II and stage III CRC
phocytes with various biological functions.67,68 In CRC, Pang and tissues by immunohistochemistry techniques and noted that posi-
colleagues69 reported that CD26þ cells have more adhesion ten- tive expression of CD24 was correlated with the poor survival of
dency to fibronectin and type 1 collagen in comparison with patients with CRC. On the other hand, Ahmed and colleagues87

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 Cancer Stem Cells in Colorectal Cancer
Table 2 Cancer Stem Cell Markers for the Prognosis of Colorectal Cancer

Expression in Normal or Role in Prognosis of

Name of Marker Noncancer Stem Cells Function Colorectal Cancer References
ALDH1 Several tissues and highest in the Detoxifying enzyme and responsible Overexpression is associated with 53,71-73
liver for oxidation of intracellular aldehydes cancer release, distant metastasis,
higher cancer grade and poor
patient survival
CD24 B-lymphocytes and differentiating Cell adhesion molecule Increased expression is correlated 82,84,86
neuroblast with poor patient survival
CD44 Epithelial cells Cell surface glycoprotein and involved Decreased or loss of expression is 53,85,88,90,91
in cell adhesion and migration, correlated with poor patient survival
participate in malignant progression
(adenoma to carcinoma)
CD133 Stem cells in different organs Regulation of stemness, associated Elevated expression at protein and 35,41,97-99
with primitive cells and mRNA level is associated with poor
transmembrane glycoprotein patient survival
CD166 Activated T cells, fibroblasts, neurons, Cell adhesion molecule, involved in Irregular and overexpression is 53,109
activated monocytes and neuronal extension, embryonic associated with shortened patient
melanoma cells hematopoiesis, embryonic survival
angiogenesis, and associated
with the development of adenoma
to carcinoma
EpCAM Epithelial tissue, progenitor cells and Cell adhesion, participate in Reduced expression is associated 53,55,113,117
stem cells Cadherin-Catenin and Wnt pathway with lymph node metastasis,
infiltrating tumor margin, higher
cancer grade, vascular invasion,
distant metastasis and poor
patient survival
Lgr-5 Adult stem cells, muscle, placenta, Associated with intestinal stem cells Higher expression is associated with 35,37,119,120,122
spinal cord, and brain and downstream target of Wnt lymph node metastasis, distant
pathway metastasis and poor patient survival
Nanog Embryonic stem cells and Transcriptional regulator, self-renewal Elevated expression is associated 24,123
epithelial cells with lymph node metastasis and
poor patient survival
SOX-2 Embryonic stem cells, neuronal Transcription factor and regulates Overexpression is correlated with 35
cells in the stomach and central self-renewal or pluripotency of recurrence and lower disease-free
nervous system undifferentiated cells survival
Oct 4 Stem cells in different organs Regulation of stemness Expression is negatively correlated 23
with cancer depth, lymph node
metastasis, and lymphatic invasion

examined whole tissues sections of colorectal adenoma (n ¼ 10) and can be an independent prognostic factor for advanced CRC, espe-
CRC tissue microarray samples from 345 patients using immuno- cially in stage IV disease. In addition, Choi and colleagues85 re-
histochemistry and did not find the prognostic implication of CD24 ported that CD44 expression was significantly correlated with
in patients with CRC. In this study, positive immunoreactivity was tumor size in patients with colorectal adenocarcinoma (n ¼ 523).
noted in 90% (9/10) of colorectal adenoma and 91% (313/345) of Furthermore, Ngan and coworkers90 demonstrated that loss of
CRC tissue samples. This lack of association with CD24 and pa- CD44 protein expression in CRC tissue samples (n ¼ 140) in
tient outcome in CRC might be attributed to the poor represen- immunostaining strongly correlated with poor survival and indi-
tation of cancer cells in the tissue microarray sections. Taken cated that CD44 loss has worse impact on patients’ prognosis.
together, more research with large number of CRC tissue samples as Despite all the positive correlations noted, Morrin and Delaney91
well as functional studies are imperative to establish the prognostic examined CD44v6 protein and mRNA expression by immunohis-
value of CD24 in CRC. tochemistry and reverse transcriptase polymerase chain reaction
(RT-PCR) in 88 CRC tissues and found no correlation of CD44v6
CD44 protein and mRNA expression with cancer stages, grade, differen-
Huh and colleagues88 demonstrated that CD44 was expressed in tiation, or survival of the patients. These conflicting results might be
100% (74/74) of CRC and its expression was significantly associ- associated with heterogeneity in cancer cells from different pop-
ated with depth of invasion and lymph node involvement. Also, ulations and varying sample sizes in the study population.
Wielenga and colleagues89 reported that CD44v6 overexpression in Furthermore, Jing et al92 noted that CD44 mRNA expression
frozen tissue sections obtained from patients with CRC could was higher in CRC metastases in liver when compared with the
identify patients who are highly predisposed to develop distant primary cancer in a cohort of 36 patients. Also, the expression was
metastasis. Furthermore, they demonstrated that CD44 expression an independent prognostic factor.

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 S.M. Riajul Wahab et al
CD133 CD166
CD133 is a known stem cell marker and is widely used as a CD166 expression has been reported to be correlated with the
marker for identifying colon CSCs.93-95 Saigusa and coworkers35 pathogenesis of various cancers, including melanoma, breast, pros-
investigated the expression level of CD133 gene and protein in tatic, esophageal, ovarian, urinary bladder, and CRCs.104-110 In
patients with rectal cancer (n ¼ 33) after chemoradiation therapy. CRC, Weichert and colleagues109 demonstrated that CD166 pro-
They noted that increased expression of CD133 both in gene and tein expression, as detected by immunohistochemistry, in CRC
protein level is correlated with distant recurrence and poor prog- (n ¼ 111) was significantly associated with the survival time of
nosis. Also, Kemper and colleagues41 noted that CD133 mRNA patients with CRC. They also noted that CD166 frequently upre-
expression predicted poorer survival in 90 patients with stage II gulated in CRC and can be act as independent prognostic marker in
colorectal carcinomas. CD133 mRNA was noted to higher in he- progression of the cancer.109 In addition, Horst and coworkers99
patic metastases from patients with colorectal carcinoma when studied the expression of CSC markers CD133, CD44, and
compared with the primary cancer.92 CD166 in CRC (n ¼ 110) by immunohistochemistry and noted
On protein expression level, Choi and colleagues85 studied that these CSC markers had significant prognostic implication in
CD133 protein expression in CRC tissues (n ¼ 523) by immu- the prognosis of patients with CRC. Furthermore, Sim and col-
nohistochemistry and found that there was significant relation of leagues111 examined preoperative chemoradiotherapy-treated colo-
CD133 expression with advanced T-stage cancer. Also, Jao et al96 rectal adenocarcinoma (n ¼ 112) by immunohistochemistry and
investigated the protein expression of CD133 in colonic adeno- noted that the expression of CD166 protein was correlated with
carcinoma (n ¼ 157) and rectal adenocarcinoma (n ¼ 76) tissue cancer regression and poor patient prognosis. These studies imply
samples by immunohistochemistry and noted that the cytoplasmic that CD166 is a key regulator in maintaining stemness in colon
expression of CD133 protein was significantly associated with cancer cells and it has the potential to be used as a prognostic maker
cancer local recurrence, survival, and cancer regression after con- for the clinical management of patients with CRC.
current chemoradiotherapy.
CD133 mRNA expression in liver tissues with metastatic CRC EpCAM
(n ¼ 50) as studied by quantitative real-time polymerase chain reac- The colon CSCs marker, EpCAM, has been reported to over-
tion showed that CD133 expression was significantly correlated with express in many human cancers including CRC and has an
poorer survival of patients with CRC.97 In addition, Horst and col- important role in cancer pathogenesis and prognosis.112-114 Went
leagues98 examined CRC tissue samples (n ¼ 57) by immunohisto- and colleagues113 examined colon cancer tissue microarrays (n ¼
chemistry and demonstrated that high CD133 protein expression is 1186) by immunohistochemistry and noted that high expression of
an independent prognostic factor and correlated with poor survival EpCAM was significantly associated with higher-grade CRC. Zhou
time of patients with CRC. Also, the group noted that high CD133 and coworkers115 studied the expression of EpCAM and Wnt/b-
expression correlates with synchronous liver metastasis.99 Further- catenin in colon cancer (n ¼ 50) and non-neoplastic intestinal
more, Kojima and coworkers100 studied CD133 protein expression by mucosae (n ¼ 20) by immunohistochemistry and noted higher
immunohistochemistry in CRC tissues (n ¼ 189) and reported that expression of EpCAM in colon cancer. They also reported high
high CD133 expression was associated with shorter recurrence-free EpCAM expression was related to lower survival rates of patients
survival and also with poor survival of patients with CRC. with CRC.115 On the other hand, Lugli et al53 demonstrated that
In the literature, a number of studies examined the role of reduced EpCAM expression was associated with tumor invasion,
CD133 mRNA expression in peripheral blood samples obtained lymph node metastasis, and high tumor grade. Other studies
from patients with CRC to evaluate the prognostic value of CD133 demonstrated that loss/reduced expression of EpCAM was corre-
in patients with CRC. High CD133 mRNA expression in the pe- lated with poor survival and cancer recurrence in patients suffering
ripheral blood of patients with CRC (n ¼ 100) was correlated with from CRC.73,116,117 Therefore, more studies are needed to confirm
recurrence of CRC and can be used as independent prognostic the prognostic role of EpCAM in CRC.
factor in CRC.101 In addition, Iinuma and colleagues102 studied the
expression of carcinoembryonic antigen (CEA), cytokeratins Lgr-5
(CK19, CK20), and CD133 in peripheral blood samples (n ¼ 735) Lgr-5 overexpression has been reported to play an active role in
obtained from different stages of CRC by real-time RT-PCR assay. regulating pathogenesis of CRC.118 Takahashi and colleagues40
They reported that overall disease-free survival of patients with CRC illustrated that high expression of Lgr-5 was related to lower
who are positive for CEA/CK/CD133 (especially in stage III can- disease-free survival and presence of metastases to lymph node and
cers) was significantly poorer when compared with those who were liver. Also, Liu and coworkers119 investigated Lgr-5 mRNA and
negative for CEA/CK/CD133.102 Conversely, Gazzaniga and col- protein expression in primary colon cancer tissues (n ¼ 366) and
leagues103 showed that the expression of CD133 mRNA in circu- xenograft mice tissues (n ¼ 40) by real-time polymerase chain re-
lating tumor cells isolated from peripheral blood of patients with action and immunostaining, respectively. They found that Lgr-5
metastatic CRC (n ¼ 45) had no correlation with overall outcome protein and mRNA significantly overexpressed in tissues from pa-
of the patients. tients with CRC and correlated with higher cancer stages and poorer
Despite having a conflicting single study, most of the studies patient survival.119
support the potential of colon CSCs marker CD133 as prognostic Wu and colleagues37 reported that Lgr-5 protein expression in
marker and more validation is required for its future use in clinical CRC (n ¼ 192) as detected by immunohistochemistry was signif-
setting. icantly overexpressed when compared with that of non-neoplastic

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 Cancer Stem Cells in Colorectal Cancer
mucosae. They also noted that higher expression of Lgr-5 protein Therapeutic Implication of CSCs in
was associated with higher histological grade, invasion, lymph node CRC
metastasis, distance metastasis, and poorer survival of patients with Conventional cancer therapies can eradicate the cancer mass
CRC.37 In addition, Hsu and colleagues120 demonstrated that high partly and could make the disease more aggressive through recur-
expression level of Lgr-5 was correlated with shorter disease-free rence and metastasis.125 The principal limitation of current che-
survival and shorter cancer-specific survival of patients with CRC. moradiotherapy is that it only eliminates differentiated cancer cells
They reported that patients with low expression of Lgr-5 showed but is insensitive to the CSCs.126 CSCs are the population of cancer
better response than patients with higher expression of Lgr5 toward cells that are responsible for therapy resistance, cancer relapse, and
5-fluorouracilebased treatment. Furthermore, Saigusa and col- distant metastasis.126,127 These phenomena in turn confer more
leagues121 demonstrated that Lgr-5 expression was highly expressed complications to the patients in the course of disease. Thus, the
in specimens obtained from patients with poor pathological development of treatment modalities targeted both conventional
response and cancer recurrence. They also found that patients with cancer cells and CSCs has greater translational implication in the
higher expression of Lgr-5 showed a significantly lower recurrence- clinical setting for the better management of cancer.
free survival. The identification of putative CSC markers and the underlying
A meta-analysis carried out by Han and coworkers122 revealed signaling pathway they involved are critical for the development of
that Lgr-5 overexpression was correlated with poor survival in pa- novel therapeutic approaches. Also, the drug-induced toxicity would
tients suffering from CRC. Overall, Lgr-5 is proposed to be an be minimized by developing therapies targeting specific molecules
efficient prognostic marker for patients with CRC. or the pathways that are active in CSCs.128 To achieve these goals,
the prospective therapeutic strategies to specifically target CSCs that
Nanog are under development include (1) the eradication of CSCs by
Xu and colleagues123 examined Nanog mRNA and protein targeting selective markers expressed on the CRC CSCs, and (2) the
expression in CRC (n ¼ 360) by real-time polymerase chain reac- inhibition or interference of CSC-specific pathway (Figure 1).
tion assay and immunohistochemistry and the expressions were
correlated with high histological grade, advances cancer stages, and Colon CSC Eradication Targeting
presence of lymph node and liver metastases in patients with CRC. Cell Surface Markers
Also, Meng and colleagues24 found that higher expression of Nanog Monoclonal antibodies/immunotoxins specific for the cell surface
was associated with shorter survival or recurrence-free survival. molecules of CSCs have the potential to eliminate the target CSC
Their meta-analysis also showed that Nanog is a potential inde- selectively.129,130 It was demonstrated that the therapeutic agents
pendent prognostic factor of the outcomes of patients with CRC. targeting cell surface markers (eg, CD133, CD44, CD26, CD29,
Nevertheless, Saiki and coworkers33 described that there was no EpCAM) could potentially eliminate CSCs, which in turn has the
correlation of Nanog mRNA expression with clinicopathological capacity to repress tumor size, reduce the metastatic potential
parameters of CRC (n ¼ 79). Thus, more studies with a large of cancer cells, and decrease cancer cell resistance to chemo-
number of samples are needed to establish the prognostic role of therapy.131-135 For example, CD133þ colon CSCs exhibited resis-
Nanog in CRC. tance to the conventional chemotherapeutic agents (eg, 5-fluorouracil
and oxaliplatin) by increased secretion of cytokine IL-4 and escaped
SOX-2 the apoptotic insults caused by the treatment.134,136 Importantly,
Saigusa and colleagues35 examined the expression pattern of colon cancer cells treated with 5-fluorouracil, oxaliplatin, and
SOX-2 both at mRNA and protein level in 33 patients with rectal monoclonal antibodies to IL-4 remarkably augmented the antitumor
cancer after chemoradiation therapy by RT-PCR and immunohis- activity of the treatments.134,136 Dallas and colleagues132 reported
tochemistry. They found that both the mRNA and protein for that chemoresistance fraction (CD133þ and CD44þ) of HT29
SOX-2 are overexpressed in all these patients. They also noted that CRC cells showed increased expression of type 1 insulinlike growth
higher expression of SOX-2 is correlated with poor disease-free factor receptor (IGF-IR). Treatment of these therapy-resistant cells
survival and distant recurrence.35 Also, Lundberg and col- with IGR-IR monoclonal antibody caused significant inhibition of
leagues124 noted that the expression of Sox-2 of 441 CRC by tumor growth in murine xenograft model.132 In addition, treatment
immunohistochemistry and noted that SOX-2 was expressed in of patients with stage III CRC (n ¼ 189) with monoclonal antibody
11% of the CRC and the expression was related to BRAFV600E against EpCAM (colon CSC marker) improves cancer-free survival
mutation. SOX-2 expression was noted in the liver metastases of the and prolongs cancer remission in patients with CRC.137
patients with SOX-2epositive CRCs. Studies demonstrated that monoclonal antibodies specific for
CD24 cell surface marker significantly inhibited the colon cancer
Oct 4 growth and tumorigenic potential both in vitro and in vivo in a
Matsuoka and colleagues23 demonstrated that Oct3/4 was mouse model.133 Also, downregulation of CD24 expression using
expressed in 129 (44%) of 290 gastric cancers and noted the cor- short hairpin RNA (shRNA) retarded tumorigenicity in human
relations of the protein expression with prognosis of patients with cancer cell lines in culture and athymic mice.133
gastric cancers. In CRC, Saigusa and colleagues35 reported higher Downregulation of CD29 by antisense oligonucleotide inhibited
expression of Oct 4 in patients with rectal cancer after treatment human colon cancer cell (HT29) migration in vitro and hepatic
with chemoradiation (n ¼ 33) was correlated with poor survival and metastasis in vivo.131 Park and colleagues131 reported that barberine
distant recurrence. (an alkaloid natural product) inhibited the migration of human

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 S.M. Riajul Wahab et al
Figure 1 Implication of Cancer Stem Cells (CSCs) as a Therapeutic Target in Colorectal Cancer Treatment. Metastatic Cancer Stem
Cells Can Be Metastasized to Another Organ and Lead to the Formation of New Cancer. The Treatment With CSC-Specific
Therapy Can Eradicate All the CSC Population. The Other Cancer Cells Can Be Destroyed by the Immune System or
Conventional Therapies. Treatment With CSC-specific Therapy Can Kill All the CSC Cells. The Rest of the Cancer Mass Can Be
Eradicated With Conventional Chemo/Radiotherapy. Treatment With Conventional Therapy Cannot Destroy the CSC
Population due to Their Resistance Mechanism and Relative Quiescence State. This May Lead to the Formation of New
Cancer. A Combined Approach Including CSC-Specific Therapy as Well as Conventional Therapy Could Fully Eradicate the
Cancer

colon cancer cells (HCT116 and SW-480) by reducing CD29 was demonstrated that inhibition of these pathways by chemical
(integrin b 1) expression. They noted that barberine treatment in- intervention increased the sensitivity of CSCs to chemotherapy.128
duces AMP-protein kinase signaling pathways in colon cancer cells, g-secretase inhibitors have the potential to inactivate Notch
which in turn reduce the CD29 protein level and decreased the signaling and can be used to develop therapeutic strategies for the
phosphorylation of CD29 targets.131 In addition, Kanwar et al138 treatment of patients with CRC.141 Constitutive activation of Wnt/
demonstrated that treatment of human colon cancer cells with b-catenin pathways in colon cancer makes this pathway an impor-
difluorinated-curcumin in combination with conventional chemo- tant target for therapy development.141 Deregulation of this
therapy (5-flurouracil and oxaliplatin) significantly reduced the pathway by inhibiting b-catenin accumulation and/or expression,
CD44 and CD166 population. This treatment caused cancer and disrupting its interaction with other components has been re-
growth inhibition, induction of apoptosis, and disintegration of ported to reduce colon cancer growth both in vitro and in vivo in a
colonospheres.138 Therefore, therapeutic strategies targeting cell xenograft mouse model by Green and colleagues.142 They treated
surface markers of colon CSCs or their downstream signaling mice implanted with colon cancer cells (SW-480) with different
partners in combination with conventional therapy has the concentrations of b-catenin antisense oligonucleotides and they
emerging potential to efficiently manage progression of CRC. noted dose-dependent tumor growth inhibition when compared
with the scrambled control b-catenin oligonucleotides group.142 van
CSC Elimination by Targeting the de Wetering and coworkers143 reported that a small compound
Signaling Pathways called inhibitor of Wnt production (IWP) has the potential
Activation of Notch, Wnt/b-catenin, TGF-b, and Hedgehog to disrupt Wnt/b-catenin pathway by inhibiting porcupine
signaling pathways have been reported to be contributed to the (a membrane-bound acetyl transferase) activity, which is essential
chemoradiotherapy resistance of CSCs in cancer treatment.139,140 It for the production of Wnt protein.

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 Cancer Stem Cells in Colorectal Cancer
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The author thanks Griffith University for provision of the higher- 31. Basu-Roy U, Seo E, Ramanathapuram L, et al. Sox2 maintains self-renewal of
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Disclosure significance of inducing pluripotent stemness-related gene expression in colorectal
The authors have stated that they have no conflicts of interest. cancer cells. Ann Surg Oncol 2009; 16:2638-44.
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