Download Stem cells in cancer: instigators and propagators?

Commentary
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Stem cells in cancer: instigators and propagators?
Malcolm R. Alison1,*, Shahriar Islam1 and Nicholas A. Wright2
1
Centre for Diabetes, and 2Centre for Digestive Diseases, Blizard Institute of Cell and Molecular Science, Barts and The London School of
Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK
*Author for correspondence ([email protected])
Journal of Cell Science 123, 2357-2368
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.054296
Journal of Cell Science
Summary
There is growing realization that many – if not all – cancer-cell populations contain a subpopulation of self-renewing stem cells known
as cancer stem cells (CSCs). Unlike normal adult stem cells that remain constant in number, CSCs can increase in number as tumours
grow, and give rise to progeny that can be both locally invasive and colonise distant sites – the two hallmarks of malignancy.
Immunodeficient mouse models in which human tumours can be xenografted provide persuasive evidence that CSCs are present in
human leukaemias and many types of solid tumour. In addition, many studies have found similar subpopulations in mouse tumours
that show enhanced tumorigenic properties when they are transplanted into histocompatible mice. In this Commentary, we refer to
CSCs as tumour-propagating cells (TPCs), a term that reflects the assays that are currently employed to identify them. We first discuss
evidence that cancer can originate from normal stem cells or closely related descendants. We then outline the attributes of TPCs and
review studies in which they have been identified in various cancers. Finally, we discuss the implications of these findings for
successful cancer therapies.
Key words: Clonogenicity, Immunodeficient mice, Self-renewal, Stem cells, Tumour-propagating cells
Introduction
Cancer begins in normal somatic cells with a mutation that is
fixed by a round of cell proliferation – a process termed initiation.
If the mutated cell has a selective growth advantage over its
normal cell neighbours owing to, for example, enhanced
proliferation or resistance to apoptosis, then a clone of cells
carrying the same mutation will emerge; in the colon this would
manifest as a small adenoma. Further tumour progression can be
viewed as an evolutionary process: if a second mutation in one
cell of the nascent cell clone gives that particular cell a further
growth advantage, then a new clone will outgrow the preceding
clone; in the colon, this would manifest as a larger adenoma with
more cellular irregularity (dysplasia). Further rounds of mutation
and clonal expansion give rise to progressively more abnormal
clones until an additional mutation produces the malignant cell
phenotype that can invade beyond normal tissue boundaries and,
subsequently, spread to distant sites – a phenomenon known as
metastasis.
We now believe that most tumours contain a subpopulation of
malignant cells with stem cell properties. These stem cells can give
rise to new tumours when xenografted, usually in small numbers,
into immunodeficient mice – hence, we refer to them as tumourpropagating cells (TPCs). However, it is currently unclear whether
TPCs arise directly from the transformation of normal stem cells
or whether they are derived from aberrant de-differentiation of
more mature neoplastic cells at a later stage of cancer progression.
Microscopic inspection of most tumours reveals a complex
heterogeneous picture. Tumours have a hierarchical nature, in part
because TPCs can give rise to more TPCs as well as to transitamplifying cells (TACs) and terminally differentiated (TD) cells
(Fig. 1). However, phenotypic and behavioural heterogeneity is
also generated by variation within different parts of a tumour with
respect to their proximity to the vascular network, and because not
all of the cells are cancer cells. Non-cancer cells within a tumour
can include inflammatory cells, cancer-associated fibroblasts and
immature myeloid cells, all of which influence tumour behaviour
and often facilitate invasion and metastasis (Coussens and Werb,
2002; Alison et al., 2009b). The microenvironment surrounding
blood vessels is conducive to the highest rates of tumour-cell
proliferation, but it can also serve as the local stem cell niche, a
specialised microenvironment of nerves, mesenchymal cells and
extracellular matrix molecules that regulate aspects of stem cell
behaviour, particularly the choice between asymmetric division
(which underlies self-renewal) and symmetric division (which
leads to an increase in the number of stem cells) (Alison and Islam,
2009).
In this Commentary, we discuss various lines of evidence that
illustrate how stem cells are central to cancer biology. First,
mutation in the DNA of normal stem cells appears to be the
initiating event in many (although not all) types of cancer. Second,
cancers themselves have stem cells (referred to here as TPCs).
Third, TPCs can be prospectively isolated based on the expression
of specific surface markers or intracellular enzyme activity. Fourth,
TPCs can be remarkably resistant to therapies that kill the majority
of cancer cells. Fifth, there are several new and, hopefully, moreeffective therapies currently being designed to target the unique
attributes of these cells.
Normal stem and progenitor cells and the
origins of cancer
It is widely believed that tumorigenesis commonly begins in normal
adult stem cells or progenitor cells that have recently descended
from them. This concept is supported first by the fact that stem
cells have – or can re-acquire – the self-renewal mechanisms
needed for maintaining and expanding stem cell numbers. Second,
stem cells are anchored to the niche and are therefore not swept
away in the prevailing cell flux, allowing time to acquire further
mutations (Fig. 1); this is particularly pertinent to cancers of
continually renewing populations, such as cells of the
haematopoietic system, gut and skin.
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Journal of Cell Science
Small intestine
In the mouse small intestine there is both a slowly cycling stem
cell population found ~4-5 cell positions above the base of the
crypt (Sangiorgi and Capecchi, 2008), and a rapidly cycling stem
cell population composed of slender-shaped cells [so-called crypt
base columnar cells (CBCCs)] that are sandwiched between Paneth
cells at the base of the crypt (Barker et al., 2007). The stem cell
characteristics of the two compartments have been established
using elegant genetic-lineage tracking techniques. The former cell
population expresses Bmi1, a component of the polycombrepressing complex 1 (PRC1) that prevents stem cell senescence;
this Bmi1-expressing population can give rise to clones that contain
all intestinal lineages. CBCCs, by contrast, express the Wnt target
gene Lgr5, which encodes an orphan G-protein-coupled receptor
(Barker and Clevers, 2010); these Lgr5-expressing cells are also
multipotential stem cells (Barker et al., 2007). Targeted deletion of
the tumour suppressor gene Apc (adenomatous polyposis coli) in
Lgr5-expressing CBCCs resulted in rapidly growing adenomas,
whereas targeted deletion of Apc in the higher-positioned TACs
that are the direct descendants of either stem cell population failed
to induce substantial adenoma growth (Barker et al., 2009). These
data illustrate that a mutation in a stem cell population is most
effective for tumour initiation in the short term. However, small
adenomas derived from TACs persisted for nearly a year in this
model, so the possibility that cancer later arises from these
adenomas cannot be excluded, particularly given the fact that
human colorectal cancers grow very slowly (Camplejohn et al.,
1973). Intriguingly, 36 days after induction of the stem-cell-derived
adenomas, both colonic and small intestinal adenomas contained
~6.5% Lgr5-positive (Lgr5+) cells; it would be interesting to know
whether these cells become TPCs. The expression of Lgr5 in mice
can occur in the same cells that express prominin 1 (PROM1, also
known as CD133, a family member of glycosylated pentaspan
membrane proteins; Box 1). Activation of Wnt signalling
specifically in these CD133-positive (CD133+) cells demonstrated
that all neoplastic tissue came from CD133+ cells (Zhu et al., 2009).
Remarkably, the frequency of CD133+ cells in the adenomas (7%)
was the same as the frequency of Lgr5+ cells in the adenomas
generated in the study in which Apc was deleted from the Lgr5expressing population. Intestinal tumours may also be initiated
from the stem cell population at positions 4-5 above the base of
the crypt. These cells overexpress Wip1 phosphatase, an enzyme
that turns off the DNA-damage response through inactivation of
stress-induced kinases such as Chk2. ApcMin/+ mice are a model
of familial adenomatous polyposis; the absence of Wip1 in ApcMin/+
Wip1-null mice severely reduces adenoma burden through p53dependent apoptosis of damaged stem cells because the DNAdamage response is extended (Demidov et al., 2007). The absence
of the hyaluronan receptor CD44 also reduces adenoma burden in
ApcMin/+ mice, probably because of increased apoptosis of initiated
cells. As CD44 is expressed only by CBCCs and early TACs in
intestinal tissues, this further supports the idea that these cells can
initiate adenomas (Zeilstra et al., 2008).
Prostate
It was previously thought that all tumours in the human prostate
gland arose from androgen receptor (AR)-negative (AR–) basal
stem cells (Maitland and Collins, 2008). Indeed, androgen inhibition
does not reduce the clonogenicity of prostatic cancer cell lines
but does reduce the size of individual colonies, consistent with the
finding that androgens are unnecessary for the maintenance of
Box 1. Defining markers of human TPCs
ALDH
The ALDH gene superfamily encodes detoxifying enzymes; high
expression of ALDH can be detrimental to tumour eradication (Dylla et
al., 2008).
Bmi1
Polycomb protein; suppresses the INK4A and ARF genes.
CD24
A sialoglycoprotein that acts as a ligand for P-selectin. It enables cells
to bind to platelets – tumour-platelet thrombi protect cells in the
bloodstream and in turn facilitate tumour invasion through interacting
with endothelia.
CD44
A transmembrane glycoprotein that binds hyaluronan (HA); often
expressed in various isoforms.
CD90
Also known as Thy-1; a glycosylphosphatidylinositol-anchored protein.
CD105
A type I integral membrane protein.
CD117
Also known as c-kit; a tyrosine kinase receptor for SCF.
CD133
The first identified member of the prominin family of pentaspan
membrane proteins (Mizrak et al., 2008). It shows restricted expression
in plasma-membrane protrusions such as epithelial microvilli (Bauer
et al., 2008). Two antibodies – CD133/1 (also referred to as AC133) and
CD133/2 (also referred to as AC141) – recognize different glycosylated
epitopes; most studies use CD133/1. CD133 expression is influenced by
cell-cycle status (CD133+ cells are generally in cycle) (Sun et al., 2009;
Jaksch et al., 2008; Tirino et al., 2008). In gliomas, the expression of
HIF2 correlates with expression of CD133; thus, hypoxia might block
TPC differentiation (Li et al., 2009).
CD166
Activated leukocyte cell-adhesion molecule (ALCAM).
EpCAM
A glycosylated type I integral membrane protein expressed by many
tumour cells (Munz et al., 2009). Involved in components of Wnt
signalling that stimulate cell proliferation after cleavage of EpCAM
intracellular domain.
SP
Side population; defines cells that are fluorescent-dull by flowcytometric analysis. SP cells efflux fluorescent dyes, such as Hoechst
33342 and DyeCycle Violet, which is a phenotype that usually depends
on expression of the ABC superfamily of membrane transporters. The
49 human ABC transporters are organized into seven families (A-G;
http://www.nutrigene.4t.com/humanabc.htm). In particular, ABCG2
(also known as breast cancer resistance protein 1, BCRP1) is expressed
in many stem cells and is upregulated in hypoxic environments (such as
stem cell niches) (Teramura et al., 2008).
stem cells but required for proliferation of TACs (Bisson and
Prowse, 2009). However, in some human prostate cancer cell lines,
CD133+ TPCs are also AR-positive (AR+), suggesting that they
originated from differentiated luminal cells (Vander-Griend et al.,
2008). Likewise, in mice, a rare population (<1% of total) of
castration-resistant luminal cells that expresses the transcription
factor Nkx3-1 (a negative regulator of prostatic epithelial
proliferation) was found to have self-renewing and bipotential
properties. Furthermore, targeted deletion of the phosphatase Pten
Stem cells in cancer
(phosphatase and tensin homologue; a known tumour suppressor
in several contexts) in these cells after androgen-stimulated
regeneration of the epithelium promoted the rapid development of
prostatic intraepithelial neoplasia (PIN) and microinvasion (Wang
et al., 2009). Thus, in the prostate, there might be two distinct cell
types from which tumours arise – basal and luminal. In particular,
the indication that these cancer-initiating cells are of luminal origin
is consistent with the absence of basal cells in human prostatic
adenocarcinoma.
Journal of Cell Science
Mammary and gonadal tissues
There might also be more than one cell type in breast tissue from
which cancer develops. Many breast cancers are believed to
originate from undifferentiated oestrogen-receptor-negative (ER–)
multipotential basal stem cells, the numbers of which can be hugely
expanded by exposure to progesterone (Joshi et al., 2010), perhaps
explaining why more reproductive cycles (early menarche, late
menopause) are a strong risk factor for breast cancer. However, in
basal-like breast cancers, gene-expression profiling suggests that
tumours originate from luminal progenitors. Moreover, luminal
progenitors are expanded in pre-neoplastic tissue from carriers of
mutations in the breast cancer 1 gene (BRCA1) (Lim et al., 2009).
Two distinct cell types (one enriched for myoepithelial transcripts)
that were differentiated from human breast epithelium and
transfected with the same defined genetic elements produced very
different tumour types (Ince et al., 2007). This observation suggests
that clinical differences between the various subtypes of breast
cancer reflect different populations of founder cells. In the testis,
pluripotent germ-cell tumours (teratomas) probably arise from
maturation-arrested gonocytes (foetal germ cells), which are
phenotypically similar to the probable precursor lesion of teratomas,
known as carcinoma in situ (CIS) (Kristensen et al., 2008); the
expression of stem cell factor (SCF) detects CIS cells (Stoop et al.,
2008).
Liver
Two distinct cell populations can give rise to hepatocellular
carcinoma (HCC). Because oncogenic transgenes can be driven
by albumin promoters, hepatocytes are implicated in many animal
models of HCC. However, hepatic progenitor cells (HPCs) that
arise from a potential stem cell compartment in the small biliary
ducts proliferate in response to chronic liver damage such as
cirrhosis, which usually precedes HCC. Therefore, HPCs might
be particularly susceptible to mutation (Alison et al., 2009a),
and maturation arrest of HPCs might contribute to tumorigenesis
in the liver. Four prognostic subtypes of HCC have been
identified that correspond to a hierarchy of liver-cell lineages
(Yamashita et al., 2008). Patients with tumours comprising a
sizeable proportion of either foetal-like liver cells or HPCs had
the poorest prognosis, whereas patients with tumours composed
of cells resembling either mature hepatocytes or biliary cells had
a more favourable clinical outcome. Moreover, gene-expression
profiling identified a subset of HCCs with a poor prognosis and
in which patient tumour-cell profiles suggested an HPC origin
(Lee et al., 2006). Finally, identifying tumour cells on the basis
of either high or low expression of the HPC marker cytokeratin
19 (CK19, also known as KRT19) can be used to identify an
HCC patient group with a shorter time to recurrence – that is,
high CK19 expression is predictive of local or distant recurrence
after whole-liver resection and transplantation (Durnez et al.,
2006).
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Lung
The phenotypic diversity of lung tumours, which vary depending
on their location, is probably due to region-specific variation in
stem cells in the tracheobronchial tree (Alison et al., 2009c). The
idea that pro-oncogenic stem cell niches exist in the lung is
supported by mouse models in which tissue-wide knockdown of a
tumour suppressor gene (e.g. p53) or upregulation of a protooncogene under the regulation of a widely expressed lung-specific
promoter (in effect inducing a ‘field cancerization’ effect), does
not induce a wide distribution of tumours. Instead, tumours are
geographically linked to stem cell niches. For example, mutations
in KRAS are common in human non-small-cell lung cancer
(NSCLC) (Riely et al., 2009), but mouse models that have
widespread Kras mutations only exhibit adenocarcinoma in the
bronchioalveolar region (Jackson et al., 2001) that involves the
expansion of self-renewing, multipotent bronchioalveolar stem
cells at the bronchioalveolar duct junction (Kim et al., 2005).
Furthermore, human small-cell lung cancers (SCLCs) arise from
mid-level bronchioles where neuroendocrine precursors are located,
whereas squamous-cell carcinomas (SCCs) generally occur in the
proximal airways and appear to develop from cytokeratin-14positive basal cells found either in the submucosal gland ducts or
intercartilagenous boundaries (Barth et al., 2000).
Haematopoietic cells
The cell of origin of most haematological malignancies is
probably a multipotential haematopoietic stem cell (HSC). For
example, in chronic myeloid leukaemia (CML), in which 95% of
affected patients have the Philadelphia chromosome (which is
likely to be the founder mutation), the BCR-ABL oncogenic fusion
transcript can be found in otherwise normal mature blood cells
that have differentiated from an HSC carrying this translocation
(Cobaleda et al., 2000). However, leukaemias might develop
from more committed progenitors that have reacquired the stem
cell property of self-renewal (Passegue et al., 2003; Dalerba
et al., 2007a). This idea is supported by the finding that
transfection with oncogenic fusion genes such as MOZ-TIF2 can
confer granulocyte-monocyte progenitors (GMPs) with leukaemic
properties (Chan and Huntley, 2008). Other examples in which
leukaemia is derived from progenitors include CML patients
in blast crisis (the final phase in the evolution of leukaemia), in
which GMPs can become self-renewing TPCs; this change is
associated with elevated Wnt signalling in the GMPs (Jamieson
et al., 2004). Likewise, in childhood acute lymphoblastic
leukaemia, clonal T-cell receptor rearrangements have been
reported to occur in CD133+, CD19+ TPCs, suggesting dedifferentiation of a cell that has already gone through the first
stages of differentiation (Cox et al., 2009). Recent studies in
mice have also highlighted the importance of the niche for stem
cell homeostasis, showing that stromal cell dysfunction can lead
to leukaemogenesis (Raaijmakers et al., 2010).
Overall, experimental and clinical evidence support the idea
that, in humans, the process of tumorigenesis begins in an adult
stem cell, although other more committed cells, particularly in the
haematopoietic system, might also be founder cells of malignancy.
Whether the cells that are operationally referred to as TPCs (on the
basis of characteristics determined in an in-vivo assay, see Fig. 2
and discussion below) are directly derived from asymmetric or
symmetric division of mutated adult stem cells, or whether they
evolve from further-differentiated cancer cells is presently unclear.
In the following sections, we explore the characteristics of TPCs
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and highlight the unique characteristics that differentiate them
from normal stem cells.
Characteristics of TPCs
Journal of Cell Science
Functional assays for TPCs
TPCs are believed to self-renew and to give rise to a hierarchy of
progenitor and differentiated cells, albeit in an unorthodox manner
that gives rise to more TPCs through symmetric divisions (Boman
et al., 2007; Cicalese et al., 2009) (Fig. 1). Operationally, TPCs are
a population of cells that can be defined by the expression of
specific molecules (see Box 1), and that are more tumorigenic than
the bulk tumour population. An example of a ‘tumorigenicity
assay’ for human TPCs that reveals their tumorigenic potential is
the transplantation of human cells into either nude or non-obese
diabetic/severe combined immunodeficient (NOD/SCID) mice,
which lack major elements of the immune system and therefore do
not reject the human cells (Fig. 2C). Such assays can be
complemented by two types of ex vivo assay known as
clonogenicity assays (Fig. 2A,B); these assays also seem to be
useful surrogates for in vivo TPC assays.
It was previously thought that TPCs were present in only very
small numbers, as large numbers of human tumour cells had to be
xenotransplanted into immunodeficient mice to generate tumours.
However, this might be because the human cells in this assay are
in a foreign microenvironment, as transplantation of mouse tumour
cells into mice indicates that TPCs can be quite common in some
tumours. For example, as few as ten mouse lymphoma or acute
myeloid leukaemia (AML) cells can propagate tumours when they
are transplanted into immunocompetent histocompatible mice
(Kelly et al., 2007). Furthermore, using standard immunodeficient
NOD/SCID mice, the frequency of TPCs in human melanoma was
estimated to be 1 in 106 (Schatton et al., 2008), but even single
human melanoma cells were found to form tumours in more highly
immunocompromised mice [NOD/SCID mice that were also null
for the interleukin-2 receptor -chain (IL2R)] (Quintana et al.,
2008). Clearly, estimated frequencies of human TPCs in cancer
crucially depend on the immune status of the recipient mouse.
Mouse models of cancer support the concept of TPCs
Several studies have reported subpopulations of TPCs in mouse
models of cancer, overcoming the initial skepticism of experiments
involving xenografting human cells into immunodeficient mice. In
a mouse model of HCC (involving targeted deletion of Pten in
hepatocytes), only a CD133+CD45– population was found to have
tumour-propagating activity in nude mice (Rountree et al., 2009).
In mammary tumours developing in p53-null mice, TPCs that were
able to form tumours which expressed both myoepithelial and
luminal markers had the phenotype of normal mammary-gland
stem cells (CD29highCD24high) (Zhang et al., 2008a), but their
frequency was only 1:300, supporting the notion that TPCs can be
a minority subpopulation. A subset of TPCs has also been identified
in mouse medulloblastomas occurring in Ptc+/– mutant mice (Read
et al., 2009).
EMT can generate TPCs
Epithelial-to-mesenchymal transition (EMT) is a process by which
the largely E-cadherin (CDH1)-dependent cell contacts between
contiguous epithelial cells break down, resulting in epithelial cells
becoming fibroblast-like and motile. For example, a main source
of fibroblasts in renal fibrosis is through EMT of kidney epithelial
cells, which is mediated by transforming growth factor 1 (TGF1).
Fig. 1. Tumour evolution and heterogeneity. TPCs probably originate from
normal stem cells (S, yellow), can be derived from transit-amplifying cells
(TACs, blue) but not terminally differentiated (TD) cells (green). TPCs can
undergo self-renewal by asymmetric cell division and increase in number by
symmetric divisions. TPCs might have a single identity (red outline), but
further genetic and epigenetic changes can give rise to other TPCs with a
different phenotype (TPC2, green outline). In addition, EMT might give rise to
more TPCs. This hierarchical organization, which gives rise to intratumoral
heterogeneity, might be supplemented by the clonal evolution of a population
of cells with a selective growth advantage (TAC, blue/yellow) (Odoux et al.,
2008; Shipitsin et al., 2007).
Transcription factors such as Twist and Snail downregulate CDH1
expression; accordingly, it has been shown that, when Twist or
Snail expression is upregulated in breast cancer epithelial cells,
EMT results. Remarkably, the resulting mesenchymal-like cells
acquire a breast TPC phenotype (CD44+CD24low) (Mani et al.,
2008). In ovarian cancer, transfection with Snail and Snail2 (also
known as Slug) leads to de-repression of ‘stemness’ genes,
including Nanog and KLF4, and four- to fivefold increases in the
size of a CD44+CD117+ TPC population that is more resistant to
chemo- and radiotherapy (Kurrey et al., 2009).
Undifferentiated tumours that have features of EMT and contain
large numbers of stem cells can also be induced in mouse mammary
glands by overexpressing the developmentally regulated homeobox
protein SIX1 (McCoy et al., 2009). SIX1 is more commonly
expressed in human metastatic lesions compared with primary
breast tumours. Another protein associated with aggressive breast
cancer is YB-1 (also known as YBX1). YB-1 activates the
translation of mRNAs that encode proteins such as Snail, Twist
and ZEB1, and other transcription factors that coordinate EMT
(Evdokimova et al., 2009). Although the rarity of TPCs has
hampered efforts to identify drugs that selectively kill them, the
acquisition of the breast TPC phenotype by transformed breast
cancer epithelial cells (CD44highCD24low) upon EMT has made
Journal of Cell Science
Stem cells in cancer
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cancer (CRC) that recapitulated the heterogeneity of the original
tumours when they were xenografted, exhibiting enterocytic,
neuroendocrine and goblet-cell differentiation – all from a single
cell (Kirkland, 1988; Odoux et al., 2008; Vermeulen et al., 2008).
In glioblastoma multiforme (GBM), single TPCs can generate all
three lineages (neural, astrocytic and oligodendrocytic; often in
different proportions), underscoring TPC multipotentiality
(Borovski et al., 2009). However, these observations do not
preclude the possibility that there are also different TPCs in a
tumour. In support of this idea, the centre and peripheral areas of
human glioblastomas have been found to contain two populations
of cytogenetically diverse TPCs: both were multipotential, but
they had distinct tumorigenic potentials (Piccirillo et al., 2009b)
(although they probably both derived from a common clone).
Cytogenetically diverse clones have also been found in human
breast cancer (Shipitsin et al., 2007) and metastatic colon cancer
(Odoux et al., 2008). Each tumour contained consistent (clonal)
karyotypic abnormalities, but also contained clones with unique
additional abnormalities, suggesting chromosomal instability that
could contribute to the generation of alternative TPCs – this
clonal evolutionary process gives rise to intratumoral
heterogeneity together with that of the TPC hierarchy (Fig. 1).
Indeed, chromosomal instability directly generates increased
numbers of tumorigenic side population (SP) cells (see Box 1) in
nasopharyngeal carcinoma (Liang et al., 2010).
Fig. 2. Assays for TPCs. Selected cells (e.g. CD133+ cells) can be assessed
either for their ability to form a large family of descendants, i.e. whether they
are clonogenic in an in-vitro assay for TPCs, or by their ability to initiate new
tumour growth after xenotransplantation into immunocompromised mice.
(A)Cells can be plated at low density directly onto plastic or in Matrigel and,
after several weeks, the plating efficiency (in percent) can be calculated as the
number of macroscopically visible clones per total number of cells plated; this
assay probably does not distinguish between stem and progenitor cells.
(B)Cells can be grown at low density in non-adherent conditions in semiliquid medium, the resulting spheres (e.g. neurospheres, colonospheres,
mammospheres) should generate both CD133+ and CD133– cells. The
resulting CD133+ cells can be serially passaged, generating secondary and
tertiary spheres with a cellular composition resembling that of the primary
spheres. (C)The gold-standard assay for TPCs in which the selected cells are
orthotopically (common for brain) or ectopically (e.g. subcutaneous site,
common for colon) xenotransplanted into an immunocompromised mouse.
high-throughput screening possible (Gupta et al., 2009). In this
study, short hairpin RNA (shRNA)-mediated inhibition of CDH1
was used to generate large numbers of TPCs, and the antibiotic
salinomycin was identified as a highly effective agent to reduce
their viability, clonogenicity and tumorigenicity.
The development of metastasis might involve the dissemination
of TPCs, in particular cells at the tumour margins that have
undergone EMT (Brabletz et al., 2009). This process might be
aided by the expression of chemokine receptors on TPCs, as
observed in pancreatic cancer (Hermann et al., 2007). Overall,
EMT in epithelial tumours appears to be an adverse prognostic
factor and can be associated with acquisition of a TPC phenotype.
The TPC niche
Normal adult stem cells seem to be regulated by molecular cues
that are provided by neighbouring connective-tissue cells, mainly
mesenchymal (fibroblast-like) cells and vascular cells; these stromal
cells contribute to what is known as the stem cell niche (Watt and
Hogan, 2000; Alison and Islam, 2009). TPCs can be present in and
influenced by a similar microenvironment. In both SCC (Atsumi
et al., 2008) and bladder cancer (He et al., 2009), putative TPCs
are located at the tumour-stroma interface. In colorectal cancer, the
promotion of Wnt signalling in TPCs requires co-stimulation by
hepatocyte growth factor (HGF) produced by stromal fibroblasts,
illustrating the importance of the microenvironment in maintaining
the tumorigenic capacity of cells within tumours (Vermeulen et al.,
2010). Brain TPCs identified on the basis of the expression of
nestin and CD133 have been observed congregated close to
capillaries in a niche; thus, therapeutic targeting of the vasculature
could destroy the niche as well as achieve tumour debulking
(Calabrese et al., 2007). In leukaemic mice, leukaemic TPCs
outcompete normal HSCs for their normal osteoblastic and vascular
niches and, as a consequence, normal HSCs lack support and
decline in number (Colmone et al., 2008). There is increasing
evidence that the leukaemic TPCs have maintenance requirements
similar to those of HSCs, which are provided by the niche (Lane
et al., 2009); therefore therapeutic targeting of the niche occupied
by leukaemic TPCs is an attractive yet difficult proposition. Such
strategies could include inhibiting leukaemic TPC homing and
engraftment by antibody blocking of molecules such as CD44
and CXCR4, which are expressed by tumour cells, and CXCL12
(also known as SDF-1), which is expressed by niche stromal cells
(Fig. 3C).
Are TPCs multipotential?
Are tumours heterogeneous because of distinct clones that arise
from different TPCs, or are the TPCs in a particular tumour
multipotential – similar to many normal stem cells? Clonal
populations have been derived from patients with colorectal
Markers of TPCs in human tumours
TPCs have been prospectively isolated on the basis of their
expression of particular markers – often CD133, but also celladhesion molecules, cytoprotective enzymes (e.g. aldehyde
Journal of Cell Science 123 (14)
Journal of Cell Science
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Fig. 3. Possible therapeutic targets in TPCs. Bold blocking arrows indicate points of therapeutic intervention. (A)Strategies based on targeting intracellular
pathways active in TPCs. The active Wnt, EpCAM, Hedgehog and Delta/Notch pathways have all been implicated in TPC self-renewal and proliferation, and can
be inhibited to therapeutically target these cells. (B)Strategies based on targeting molecules that are highly expressed by TPCs. Targeting ABC transporters and
ALDH activity might sensitise TPCs to currently available drugs. CD133 is another potential target (although CD133 expression can be ubiquitous in some
tissues). Manipulation of mRNA expression levels through microRNAs (miRNAs) is also a promising strategy by which to target TPCs. (C) Strategies based on
targeting molecules involved in invasion and metastasis. Blocking CXCR4-CXCL12 and CD44-hyaluronan (HA) interactions might target TPCs. Inhibiting EMT
might slow the generation of more TPCs with metastatic potential. The direct destruction of endothelial cells that maintain vascular niches also has the potential to
eliminate TPCs. -Cat, -catenin; Dsh, dishevelled; ECM, extracellular matrix; EpEX, EpCAM extracellular domain; Fz; Frizzled; GSK3, glycogen synthase
kinase 3; HH, Hedgehog; Lrp5/6, low-density lipoprotein receptor-related protein 5 and/or 6; PTC, Patched; SMO, Smoothened.
dehydrogenase, ALDH) and drug-efflux pumps (e.g. ABC
transporters) (Box 1). As this field is still evolving, there is not
yet an apparent consensus about the best marker by which to
identify TPCs in any particular cancer (Table 1). Brain TPCs
have mainly been studied in medulloblastoma, a paediatric tumour
of the cerebellum and gliomas: in this case, CD133 has been the
best marker to enrich for TPCs (Piccirillo et al., 2009a). By
contrast, there has not been a similar consensus on the best
marker for identifying TPCs in other cancers. This is particularly
true for gastrointestinal carcinomas, as several permutations and
Stem cells in cancer
2363
Table 1. Markers of TPCs in human tumours
Journal of Cell Science
Tumour
Phenotype
Reference
Acute T lymphoblastic leukaemia
CD90+CD110+ (Mpl)
(Yamazaki et al., 2009)
Acute myeloid leukaemia
CD34+CD38SSClowALDHbright
(Bonnet and Dick, 1977)
(Cheung et al., 2007)
Breast carcinoma
CD44+CD24–ALDH
ALDH
(Ginestier et al., 2007)
(Charafe-Jauffret et al., 2009)
Bladder carcinoma
SP
(Oates et al., 2009)
+
–
–
Childhood B-acute lymphoblastic leukaemia
CD133 CD19 CD38
Colorectal carcinoma
CD133+
CD133+
CD133+ and CD133–
CD44+
EpCAM+CD44+CD166+
CD44+ALDH1
ALDH1
EpCAM+ALDH1
(Ricci-Vitiani et al., 2007)
(O’Brien et al., 2007)
(Shmelkov et al., 2008)
(Du et al., 2008)
(Dalerba et al., 2007b)
(Chu et al., 2009)
(Huang, E. H. et al., 2009)
(Carpentino et al., 2009)
Endometrial carcinoma
CD133+
SP
(Rutella et al., 2009)
(Friel et al., 2008)
Ewing sarcoma
CD133+
(Suva et al., 2009)
+
(Cox et al., 2009)
Gastric carcinoma
CD44
(Takaishi et al., 2009)
Head and neck SCC
CD44+
ALDH
(Prince et al., 2007)
(Clay et al., 2010)
Hepatocellular carcinoma (HCC)
CD133+
CD44+CD90+
CD133+
(Song et al., 2008)
(Yang et al., 2008a; Yang et al., 2008b)
(Ma et al., 2007)
HCC cell lines
SP
(Chiba et al., 2008)
+
Hodgkin lymphoma
CD27 ALDH
(Jones et al., 2009)
Lung carcinoma (NSCLC)
CD133+
CD133+
SP
SP
(Chen et al., 2008)
(Eramo et al., 2008)
(Levina et al., 2008)
(Sung et al., 2008)
Lung carcinoma (SCLC)
SP
CD133+ASCL+ALDH1
(Das et al., 2008)
(Jiang et al., 2009a; Jiang et al., 2009b)
Medulloblastoma, glioma
CD133+
(Piccirillo et al., 2009a)
+
Osteosarcoma
CD133
ALDH
(Tirino et al., 2008)
(Wang et al., 2010)
Ovarian adenocarcinoma
CD44+CD117+
CD44+MyD88+
CD133+
(Zhang, S. et al., 2008)
(Alvero et al., 2009)
(Baba et al., 2009)
Pancreatic carcinoma
CD133+
CD133+
CD44+CD24+EpCAM+
(Hermann et al., 2007)
(Maeda et al., 2008)
(Li et al., 2007)
Prostate carcinoma
CD133+CD44+21+
CD44+
CD44+
CD44+CD24–
(Collins et al., 2005)
(Patrawala et al., 2006)
(Klarmann et al., 2009)
(Hurt et al., 2008)
Renal carcinoma
CD105+
(Bussolati et al., 2008)
SCC (A431)
Podoplanin+
SP
SP
(Atsumi et al., 2008)
(Loebinger et al., 2008)
(Zhang et al., 2009)
ASCL1, achaete-scute complex homologue 1; MyD88, myeloid differentiation factor 88; SP, side population; SSC, side scatter.
apparent contradictions have been proposed regarding the use of
various markers, particularly CD133 (Shmelkov et al., 2008).
High ALDH activity has been used to select for TPCs in human
breast cancer cell lines (Charafe-Jauffret et al., 2009). In primary
tumours, when TPCs are selected on the basis of the combined
expression of ALDH and CD44+CD24–/low, as few as 20 of these
cells are usually tumorigenic (Ginestier et al., 2007). Breast cancers
that contain cells with the CD44+CD24– phenotype are most
common in basal-like tumours, but not all breast cancers contain a
subpopulation of cells that have this phenotype (Honeth et al.,
2364
Journal of Cell Science 123 (14)
Journal of Cell Science
2008). Brca1-deficient mouse mammary tumours contain distinct
CD44+CD24– and CD133+ tumorigenic subpopulations (Wright
et al., 2008).
The first TPCs were recognized in acute myeloid leukaemia as
a small subset of cells that, similar to normal HSCs, had the
signature CD34+CD38– (Bonnet and Dick, 1997). Unlike studies
of solid tumours, among which there is little apparent agreement
regarding the identity of TPCs, the consensus view in
haematological malignancies is that the CD34+CD38– signature
does identify most TPCs. However, it should be noted that other
markers have also been used to identify TPCs in these types of
malignancy (see Table 1).
TPCs in tumour invasion and metastasis
If TPCs are involved in tumorigenesis, it follows that their
frequency in primary tumours is correlated with the extent of
tumour invasion and metastasis and, in turn, to patient prognosis
(their clinical outlook). The chemokine receptor CXCR4 is often
expressed by tumour cells and by TPCs in particular. Hence, these
cells can migrate along a gradient of the CXCR4 ligand, CXCL12,
which can originate from haematopoietic niches and many other
tissue sites, thereby facilitating metastasis. For example, CXCR4
is highly expressed by human endometrial cancer, whereas
CXCL12 is expressed by normal tissues; a CXCR4-neutralizing
antibody was effective in blocking the seeding of cancer cells at
ectopic sites in nude mice, highlighting the potential use of
antagonists of chemokine receptors as a therapeutic strategy
(Gelmini et al., 2009) (although it was not clear if TPCs were
specifically targeted in this study). Similarly, blocking CXCR4
expression in sphere-forming cells (putative TPCs) in breast cancer
cell lines using antibodies or shRNA inhibited invasive behaviour
in vitro (Krohn et al., 2009). CXCR4-expressing CD133+ TPCs are
also directly involved in the metastasis of human pancreatic cancer
cells: when CD133+ TPCs were partitioned into CXCR4+ and
CXCR4– fractions, only the CXCR4+ cells formed metastases after
orthotopic xenografting. Notably, the metastases could be blocked
by AMD3100, a small-molecule inhibitor of CXCR4 (Hermann
et al., 2007).
The invasion of ALDH+ TPCs in breast cancer cell lines involves
interleukin-8 (IL-8) and its receptor CXCR1; these TPCs expressed
high levels of CXCR1 and showed markedly enhanced invasion
through Matrigel in response to IL-8 (Charafe-Jauffret et al., 2009).
The expression of various integrins and of the transmembrane
glycoprotein CD44 on gastric cancer SP cells can also be important
for metastasis (Nishii et al., 2009).
In the metaplastic type of breast cancer that is characterised by
aggressive growth, chemoresistance and poor patient outcome, the
gene signature of the tumour generally closely matches that of
CD44+CD24–/low TPCs found in most human breast tumours
(Hennessey et al., 2009). As well as illustrating the link between
TPCs and metastatic tumours, these data also highlight that
stemness traits are usually a poor prognostic indicator.
TPCs and patient prognosis
Overall, we believe that, for a given tumour type, a high number
of stem cells indicates a poor prognosis. In breast cancer, for
example, the most poorly differentiated tumours have the highest
burden of TPCs (Pece et al., 2010). In brain tumours, high CD133
expression is a prognostic marker for reduced time of diseasefree survival and overall survival, a measure that is independent
of tumour grade, the extent of resection and the age of the patient
(Cheng et al., 2009). Another study reported that combined nestin
and CD133 expression is a marker of poor prognosis (Zhang
et al., 2008b). Neurosphere formation (an assay to identify TPCs)
has also been found to be an independent predictor of death in
patients with malignant gliomas, suggesting that the ability to
propagate neurospheres in culture is a clinically relevant
parameter (Laks et al., 2009). High CD133 expression in
colorectal carcinoma has also been found to be an independent
marker of poor prognosis (Horst et al., 2009). In pancreatic
cancer, in which 60% of tumours express CD133 (with <15%
positive cells per case), CD133 expression was found to be an
independent adverse prognostic factor for 5-year survival. In
addition, CD133 expression was significantly associated with
lymph-node metastases (Maeda et al., 2008). However, despite
the large body of evidence showing that TPCs in a wide range of
tumour types express CD133, the functional role of this molecule
in TPC activity is not clear. Although high CD133 expression is
tightly correlated with colorectal liver metastasis, small interfering
RNA (siRNA)-mediated knockdown of CD133 expression in
cultured colon cancer cell lines affected neither overall cell
migration nor invasion (Horst et al., 2009). High ALDH
expression in tumours has also been associated with poor
prognosis in a number of tumour types including breast (Morimoto
et al., 2009; Tanei et al., 2009), pancreas (Rasheed et al., 2010)
and early-stage lung cancer (Jiang et al., 2009a). High activity
levels of the ABC transporters have also been reported to be a
sign of poor prognosis; in patients with AML, there is reduced
overall 4-year survival in patients with tumours expressing high
levels of ABCC11 (also known as MRP8) (Guo et al., 2009). In
summary, patients with tumours that express high levels of
molecules associated with TPCs have a poorer prognosis than
patients with tumours that express low levels of these markers.
Therapeutic targeting of TPCs
If TPCs are the roots of cancer, then these are the cells that must
be specifically eliminated for a successful therapy. However, it is
well known that many cancers are resistant to currently available
therapies. For example, CD133+ TPCs in glioblastomas are highly
resistant to irradiation owing to upregulation of the DNA-damage
response and can be sensitised by inhibition of Chk1 and Chk2
(Bao et al., 2006), whereas the entire pool of CD133+ TPCs can be
reduced by forcing them to differentiate into astrocytes with bone
morphogenetic proteins (Piccirillo et al., 2006). Therapy can also
enrich for TPCs in the residual tumour, as seen with gemcitabine
treatment of pancreatic cancer (Mueller et al., 2009),
cyclophosphamide treatment of colorectal cancer (Dylla et al.,
2008), doxorubicin and fluorouracil treatment of HCC (Ma
et al., 2008) and cisplatin, doxorubicin and methotrexate treatment
of lung cancer (Levina et al., 2008). Therefore, highly specific
therapies must be developed to target TPCs.
TPCs and other cancer cells express several potential therapeutic
targets (Fig. 3). Targeting key signalling pathways that are active
in TPCs is one approach to therapy (Fig. 3A): for example,
inhibiting the release of Notch intracellular domain (NICD) with
-secretase inhibitors, and by blocking the Hedgehog and Wnt
pathways with cyclopamine and DKK1, respectively. A new
approach to antagonize Wnt signalling has been to stabilize axin,
thereby preserving the -catenin destruction complex (Huang,
S. M. et al., 2009). Components of the Wnt signalling pathway
also link up with the intracellular domain of EpCAM (EpICD) to
promote cell proliferation; thus, similar to the Notch pathway,
Journal of Cell Science
Stem cells in cancer
targeting the proteases involved in intracellular cleavage are
promising approaches (Munz et al., 2009).
Small-molecule inhibitors of various other molecules expressed
by TPCs is an alternative avenue for therapy (Fig. 3B). As the drug
resistance of TPCs can often be directly attributed to the activity
of ALDH (Dylla et al., 2008) or ABC transporters (Loebinger
et al., 2008; Sung et al., 2008), these are both promising targets for
small-molecule therapy (Fig. 3B). In addition, given that many
types of cancer cells have a specific microRNA-expression profile
(Nicoloso et al., 2009), the use of microRNA-based therapeutic
tools to target TPCs is an area of increasing interest. For example,
low expression of microRNA-199b-5p is a marker of poor patient
survival in medulloblastoma: this miRNA affects the Notch pathway
by downregulating Hes1 expression, and its introduction reduces
proliferation and engraftment of CD133+ medulloblastoma TPCs
in athymic mice (Garzia et al., 2009). In GBM, transfection of
microRNA-124 and microRNA-137 causes cell-cycle arrest and
apparent differentiation of CD133+ cells (Silber et al., 2008).
MicroRNA-128 is markedly downregulated in glioblastomas, and
introducing microRNA-128 into glioma cells reduces their
proliferation by directly binding to the 3⬘-untranslated region of
BMI1 mRNA (Godlewski et al., 2010).
Other therapeutic strategies might involve inhibiting invasion
and metastasis by blocking integrins or CD44 (which binds to
hyaluronan) at the cell surface (Fig. 3C). For example, CD44+
ovarian tumour cells that express markers of activated pluripotent
stem cells might have a selective advantage for spreading through
adhering to the hyaluronic-acid pericellular coat of surrounding
mesothelial cells (Bourguignon et al., 2008). Finally, smallmolecule inhibitors of CXCR4 were shown to successfully block
the metastasis of xenografted human pancreatic cancer (Hermann
et al., 2007).
As discussed above, EMT of cancer cells can generate moreinvasive cells and give rise to new TPCs; therefore, developing
therapies that inhibit this process is an important aim. EMT is a
prominent feature of basal-like breast cancer; here, inhibiting Wnt
signalling was shown not only to block stem cell self-renewal but
also represses the expression of the CDH1 repressors Twist and
Slug, which, in turn, blocks metastasis (DiMeo et al., 2009).
Conclusions and perspectives
Normal adult multipotential stem cells are self-renewing. If
normal adult stem cells are the founder cells of many tumours,
TPCs probably inherit many of the attributes of normal stem
cells. We believe that most human tumours – both solid tumours
and haematological malignancies – have a subpopulation of TPCs,
although recent data challenge the once-popular belief that they
are always a minority subpopulation. For example, in malignant
melanoma, they can make up 25% of the total cell population.
The fact that mouse tumours also contain subpopulations that are
enriched for TPCs supports the notion that malignancies contain
specific cells with stem cell attributes. Collective evidence
indicates that markers such as CD133, CD44 and ALDH are
characteristically expressed by TPCs, and that this population
can be expanded within tumours by both symmetrical division of
TPCs and/or through other cancer cells undergoing EMT. Many
therapeutic approaches are on the horizon by which to target
TPCs in cancer, which is a challenging prospect given that these
cells seem to be particularly resistant to current therapies.
2365
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