Colorectal
cancer (CRC) is the third most common form of cancer and the second cause of
cancer-related death in many industrialized countries and is characterized by a
heterogenic pool of cells with distinct differentiation patterns, leading to
655,000 deaths worldwide per year (1). Despite the emergence of new targeted
agents and the use of various therapeutic combinations, none of the treatment
options available is curative in patients with advanced cancer. A growing body
of evidence is increasingly supporting the idea that human cancers can be
considered as a stem cell disease. According to the cancer stem cell model,
malignancies originate from a small fraction of cancer cells that show
self-renewal and pluripotency and are capable of initiating and sustaining
tumor growth (2).
This
review will focus on the biology of normal and malignant colonic stem cells,
which might contribute to our understanding of the mechanisms responsible for
tumor development and resistance to therapy. First, we will briefly revise the
knowledge available on normal intestinal stem cells and recent advances in
understanding crypt biology, which have led to new theory on the origins of
colon adenomas and cancers. Then, we will summarize the evidence and current
status on colon cancer stem cells, focusing on their relevance and promises for
the treatment of colorectal carcinoma.
Colonic stem cell identification
The
adult colonic epithelium has a well-defined architecture organized into crypts,
dynamic structures which are constantly self-renewing (3). Each crypt unit is
maintained by adult multipotent stem cells (SCs), located at the bottom of the
structure itself, that are able to simultaneously self-renew and generate a
population of transiently amplifying cells which in turn generate more the
specialized intestinal epithelial mature cells. Three differentiated cell types
mediate the function of colonic epithelium: the colonocytes, also termed
absorptive enterocytes, the mucus-secreting goblet cells and the hormone-secreting
enteroendocrine cells (Figure 1).
Adult
SCs are defined by several key functional properties including: self-renewal,
potential for multilineage differentiation and tissue regeneration. Two
different models have been proposed to localize the intestinal SCs; the “+4
position” model and the “stem cell zone” model (4). According to the former,
the intestinal SCs are located at the +4 position relative to the bottom of the
small intestine crypt, just above the non-cycling Paneth cells. These cells are
quiescent, slowly cycling, label retaining cells (LRCs) and, through asymmetric
division, give rise to their differentiated progeny. The more recent “stem cell
zone” model states that active, rapidly cycling cells and self-renewal, termed
crypt base columnar (CBC) cells, are the true intestinal SCs (Figure 2). These cells are interspersed
between the Paneth cells in the small intestine or located at the very bottom
of the crypt in the colon. The intestinal stem cells, both +4 cells and CBC
stem cells give rise to the transitamplifying
population that undergoes vigorous division and differentiation into
enterocytes, goblet cells, and enteroendocrine cells as they migrate out of the
crypt onto the villi (3).
Fig. 1. Colonic crypt organization. a. In the epithelial lining of normal colonic mucosa, stem cells
(red) are located at the bottom of the crypts. Upon asymmetrical divisions, the
daughter cells undergoing differentiation migrate upward to give rise in turns
to transitamplifying (TA) precursors (light blue) and terminally differentiated
cells (pink). b. Cell types in the
colon epithelium. Intestinal stem cells generate three epithelial cell types:
the absorptive columnar cells, the hormone-producing enteroendocrine cells, and
the mucous-producing goblet cells.
Fig. 2. Models for
stem cells location of stem cells in intestinal crypts. a. The “+4
position” model suggests that intestinal stem cells are quiescent, slowly
cycling cells and located just above the
Paneth cells at position +4 relative to the crypt bottom (green). The
most important marker that identifies these cells is Bmi-1. b. The “stem
cell zone” model assumes active, rapidly cycling cells, so-called crypt base
columnar (CBC) cells (red), interspersed with Paneth cells, are
the true intestinal stem cells. The most important marker to identify these
cells is Lgr5.
The
Lgr5 gene encodes a leucine-rich repeat containing G-protein coupled
receptor, also known as Gpr49. Lgr5 expression is restricted to cycling CBC
cells and it has been demonstrated that Lgr5-expressing cells differentiate
into the expected functional lineages of the colonic epithelium (5).
Transcriptome analysis of Lgr5+ epithelial cells isolated from the bottom of
the small intestinal crypts led to the identification of a gene signature for
these Lgr5+ SCs. The OLFM-4 gene encodes a secreted molecule with unknown
function, originally cloned from human myeloblasts (6), which is enriched in
human colon crypts (7). Due to the very low expression levels of Lgr5, OLFM-4
has been recently proposed as a more faithful SC marker highly expressed in CBC
cells in human small intestine and colon (8).
Colorectal cancer stem cell identification
Tumors
are composed of a heterogeneous mixture of cancer cells at various levels of
differentiation, very similar to the structure of an organ. Recently, the
“cancer stem cell” model of tumorigenesis has proposed that within the tumor
mass there is a predetermined cell population with a ‘‘stem cell’’ phenotype,
able to perpetuate the cancer, while the rest of the tumor cells are incapable
of self-renewal (9). Even though it has long been assumed that mutations within
adult colonic stem cells may induce neoplastic transformation, the proof of
existence of colorectal cancer stem cells (CRC-SCs) has been hindered in the
past years by difficulties in identifying a specific biomarker for this rare
cell population. Only recently, new evidence has been provided that supports
the existence of CRC-SCs, confirming that the tumorigenic cell population of
CRC can be isolated on the basis of the expression of specific cell surface
biomarkers (9). The standard analysis to ascertain the existence of a
subpopulation of cancer stem cells (CSCs) is the demonstration that these cells
can transfer the tumor in immunocompromised mice and replicate the phenotypic
heterogeneity of the parental tumor. Several recent studies have evaluated the
functionality of specific CRC-SC biomarkers by using a combination of flow
cytometry to identify a “putative” SC population and xenograft models involving
immunodeficient mice to determine their tumor initiating potential (9).
In
the first two studies, CD133, also known as Prominin-1, was employed to
identify the tumorigenic cell population within CRC and metastatic (10, 11).
When transplanted into the renal capsule of NOD/SCID mice, CD133+ cells readily
developed tumors displaying morphologic features equivalent to those of the
parental cancer. Tumor phenotype was further maintained upon serial
transplantation (10). Similarly, in the second study, a population of CD133+
cells, accounting for approximately 2.5% of tumor cells, was isolated from
colon cancer specimens and perpetuated in vitro as floating colonies or “tumor
spheres” (11). CD133+ cells readily gave rise to tumors in mice,
whereas the CD133− cell population was unable to generate tumors
even after serial transplantation in mice (9). Both studies demonstrated the
expression of CD133 also in normal colon tissue, although at a lower frequency,
reinforcing the hypothesis that CD133+ CRC-initiating cells in cancer samples
might result from oncogenic transformation of normal colonic SCs.
Subsequently,
one study proposed CD44 and the epithelial surface antigen (EpCAM) as
CRC-SC-specific markers, with further enrichment by CD166. Purified CD44+/Ep- CAMHIGH
cells injected into NOD/SCID mice resulted in high frequency generation of
tumor xenograft. In contrast, CD44-/EpCAMLOW cells lack tumor
initiating activity (12). Further subfractionation of the CD44+/EpCAMHIGH
cell population by using the mesenchymal stem cell marker CD166 increased the
success of tumor xenograft.
Finally,
in a more recent study, aldehyde dehydrogenase 1 (ALDH) has been proposed as a
promising new marker for normal and malignant human colonic SCs (13). Flow
cytometric isolation of ALDH1+ cancer cells and implantation of as
few as 25 cells in NOD/SCID mice generate tumor xenografts. Further isolation
of cancer cells using a second marker (CD44 or CD133 serially) only modestly
increased enrichment based on tumor-initiating ability.
In
an another study, reported that in primary colon cancer samples from humans and
mice, CD133 was expressed in all epithelial, EpCAM+ cells in the malignant
tissue and that CD133 expression was excluded from the non-epithelial cell
components of the tumor (14). Thus, they proposed that the inability of CD133-
to generate tumors could be simply due to their non-epithelial nature.
Furthermore, the same authors demonstrated that both, CD133+/EpCAM+ and
CD133-/EpCAM+ cell populations, isolated from liver metastasis of colon cancer,
were able to generate tumors upon serial transplantation into NOD/SCID mice
(14).
A
comparison of expression of the three markers CD133, CD44 and CD166 that have
been associated with CRC-SCs revealed that the expression of CD133 correlates
with that of CD166, whereas both do not correlate with CD44, confirming that
CD133 is, alone, the best marker to predict poor patient survival (15).
Identification
of biomarkers for CRC-SCs will improve the understanding of the mechanism
underlying tumor growth and progression. Once again, studies performed on mouse
model could offer helpful suggestion. Recently, one study provided a very
convincing demonstration of the origin of intestinal cancer from Lgr5+
CBC cells. The authors have shown that deletion of APC in Lgr5+ expressing
cells leads to their transformation within days, suggesting that Lgr5 may mark
not only normal intestinal stem cells, but also a limited population of CSCs
(16). Simultaneously, using knock-in LacZ reporter mice within the
Prominin-1 (Prom1) locus, in a study have shown that Prom1+ cells,
located at the base of the crypts in the small intestine, co-express Lgr5,
generate the entire intestinal epithelium and are susceptible to neoplastic
transformation (17).
From
a clinical point of view, a recent study showed that Lgr5 was markedly
over-expressed in the majority of advanced CRCs compared with normal mucosal
tissue (18). As expected, in situ hybridization analysis confirmed the
expression of Lgr5 in CBC cells in both small intestine and colon. This Lgr5
expression, which was variable among CRC cases, correlated significantly with
lymphatic and vascular invasion, lymph node metastasis and tumor stage,
suggesting the involvement of this marker in tumor progression.
It
might be summarized the markers that have been used to isolate CRC-ICs, shown
in table 1 (9).
Table 1 Markers that have been proposed to characterize normal
intestinal SCs and used to isolate CRC-ICs
Marker
|
Function
|
|
Normal intestinal
stem cells
|
Musashi-1
|
RNA-binding
protein
|
Hes-1
|
Transcriptional
repressor
|
|
EphB
receptors
|
Cell
surface receptors
|
|
Bmi-1
|
Policomb-repressor
protein
|
|
Lgr5
|
Unknown,
WNT target gene
|
|
Aldh-1
|
Enzyme
|
|
Colon
cancer stem cells
|
CD133
|
Unknown
|
CD44
|
Hyaluronic
acid receptor
|
|
CD166
|
Adhesion
molecule
|
|
Aldh-1
|
Enzyme
|
Clinical
implications of colorectal cancer stem cells
The
CSC model has important implications for cancer therapy. At present, anticancer
therapies for CRC include surgery, radiation, chemotherapy, and anti-VEGF or
EGFR monoclonal antibodies. In most cases, many current cancer therapies target
the most rapidly dividing cells, which represent the majority of the tumor cell
population. Similarly, CRC-SCs have been found to be enriched in colon tumors
following classical chemotherapeutic regimens and remain capable of rapidly
regenerating tumor from which they were derived (19). The authors have further
demonstrated that resistance is mediated, at least in part, by ALDH1 enzyme
activity.
Together
with resistance to chemotherapy, CSCs are frequently resistant to standard
radiotherapy regimens. In this respect, it has been recently demonstrated that
resistance to radiation of CD133+ glioblastoma SCs can result from elevated
expression of DNA damage response genes (20). Radiotherapy for glioblastoma is
associated with an increase in the proportion of the CD133+ fraction.
In
another study, has recently demonstrated that the up-regulation of
interleukin-4 (IL-4) in CD133+ CRC-SCs is an important mechanism that protects
these tumorigenic cells from apoptosis. CD133+ CRC-SCs produce IL-4 as an
autocrine growth factor promoting tumor resistance to chemotherapeutic agents
such as 5-fluorouracil and oxaliplatin (21). This phenomenon was confirmed in xenografts
in which the administration of anti- IL-4 antibodies significantly reduced
tumor growth after chemotherapy.
In
a recent study, generated CD133+ tumor sphere cultures from several colon
cancer specimens and performed mass-spectrometry-based quantitative proteomics in
order to identify cell surface proteins enriched on culture tumor cells (22).
These cells retain the expression of cell surface markers such as CD133, CD166,
CD44 and EpCAM as well as other stem cell-associated proteins including nestin,
Bmi1 and Msi-1, thus confirming the value of this in vitro model for biological
analysis of CSC populations as well as for drug screening experiments.
Conclusion
Increasing
evidence shows that CRC-SCs may play a critical role in tumor development and progression.
CRC-SC resistance to conventional therapies may explain why it is difficult to
completely eradicate cancer and why recurrence is often inevitable. Thus, the
identification and molecular characterization of CSCs is critical to develop
therapeutic strategies that specifically target this rare population of cells
and that are likely to be effective in eradicating tumors and in reducing the
risk of relapse and metastasis.
References
1.
Jemal
A, Siegel R, Ward E, Murray T, Xu J,
Smigal C, Thun MJ. Cancer statistics. CA
Cancer J Clin. 2006 Mar-Apr;56(2):106-30.
2.
Boman
BM, Wicha MS. Cancer stem cells: a step
toward the cure. J Clin Oncol. 2008 Jun 10;26(17):2795-9.
3.
Radtke F,
Clevers H. Self-renewal and cancer of the gut: two sides of a coin. Science 2005;
307: 1904-09.
4.
Barker N,
van de Wetering M, Clevers H. The intestinal stem cell. Genes Dev 2008; 22:
1856-64.
5.
Barker N,
van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A,
Korving J, Begthel H, Peters PJ, Clevers H. Identification of stem cells in
small intestine and colon by marker gene Lgr5. Nature 2007; 449:
1003-07.
6.
Van der Flier LG,
van Gijn ME, Hatzis P, Kujala P, Haegebarth A, Stange DE, Begthel H, van den
Born M, Guryev V, Oving I, van Es JH, Barker N, Peters PJ, van de Wetering M,
Clevers H. Transcription factor achaete scute-like 2 controlsintestinal stem
cell fate. Cell 2009; 136: 903-12.
7.
Kosinski C,
Li VS, Chan AS, Zhang J, Ho C, Tsui WY, Chan TL, Mifflin RC, Powell DW, Yuen
ST, Leung SY, Chen X. Gene expression patterns of human colon tops and basal
crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl
Acad Sci USA 2007; 104: 15418-23.
8.
Van der Flier LG,
Haegebarth A, Stange DE, van de Wetering M, Clevers H. OLFM4 is a robust marker
for stem cells in human intestine and marks a subset of colorectal cancer
cells. Gastroenterology 2009; 137: 15-17.
9.
Ricci-Vitiani L,
Fabrizi E, Palio E, De Maria R. Colon cancer stem cells. J Mol Med 2009;
87: 1097-104.
10. O’Brien
CA, Pollett A, Gallinger S, Dick JE. A
human colon cancer cell capable of initiating tumour growth in immunodeficient
mice. Nature 2007; 445: 106-10.
11. Ricci-Vitiani
L, Lombardi DG, Pilozzi E, Biffoni M,
Todaro M, Peschle C, De Maria R. Identification and expansion of human
colon-cancer-initiating cells. Nature 2007; 445: 111-15.
12. Dalerba
P, Dylla SJ, Park IK, Liu R, Wang X, Cho
RW, Hoey T, Gurney A, Huang EH, Simeone DM, Shelton AA, Parmiani G, Castelli C,
Clarke MF. Phenotypic characterization of human colorectal cancer stem cells. Proc
Natl Acad Sci USA 2007; 104: 10158-163.
13. Huang
EH, Hynes MJ, Zhang T, Ginestier C, Dontu
G, Appelman H, Fields JZ, Wicha MS, Boman BM. Aldehyde de-hydrogenase 1 is a
marker for normal and malignant human colonic stem cells (SC) and tracks SC
overpopulation during colon tumorigenesis. Cancer Res 2009; 69:
3382-89.
14. Shmelkov
SV, Butler JM, Hooper AT, Hormigo A,
Kushner J, Milde T,et. al. CD133 expression is not restricted to stem cells,
and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. J
Clin Invest 2008; 118: 2111-20.
15. Horst
D, Kriegl L, Engel J, Kirchner T, Jung A.
Prognostic significance of the cancer stem cell markers CD133, CD44, and CD166
in colorectal cancer. Cancer Invest 2009; 27: 844-50.
16. Barker
N, Ridgway RA, van Es JH, van de Wetering
M, Begthel H, et.al. Crypt stem cells as the cells-of-origin of intestinal
cancer. Nature 2009; 457: 608-11.
17. Zhu
L, Gibson P, Currle DS, Tong Y,
Richardson RJ, Bayazitov IT, Poppleton H, Zakharenko S, Ellison DW, Gilbertson
RJ. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic
transformation. Nature 2009; 457: 603-07.
18. Uchida
H, Yamazaki K, Fukuma M, Yamada T,
Hayashida T, Hasegawa H, Kitajima M, Kitagawa Y, Sakamoto M. Overexpression of
leucine-rich repeat-containing G protein-coupled receptor 5 in colorectal
cancer. Cancer Sci 2010; Epub ahead of print.
19. Dylla SJ,
Beviglia L, Park IK, Chartier C, Raval J, Ngan L, et.al. Colorectal cancer stem
cells are enriched in xenogeneic tumors following chemotherapy. PLoS ONE 2008;
3:2428.
20. Bao
S, Wu Q, McLendon RE, Hao Y, Shi Q,
Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance
by preferential activation of the DNA damage response. Nature 2006; 444:
756-760.
21. Todaro
M, Alea MP, Di Stefano AB, Cammareri P,
Vermeulen L, Iovino F, Tripodo C, Russo A, Gulotta G, Medema JP, Stassi G.
Colon cancer stem cells dictate tumor growth and resist cell death by
production of interleukin-4. Cell Stem Cell 2007; 1: 389-402.
22. Fang
DD, Kim YJ, Lee CN, Aggarwal S, McKinnon
K, Mesmer D, Norton J, Birse CE, He T, Ruben SM, Moore PA. Expansion of
CD133(+) colon cancer cultures retaining stem cell properties to enable cancer
stem cell target discovery. Br J Cancer 2010; 102: 1265-1275.
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