INTRODUCTION
Tumour is composed of a heterogeneous group
of cells with different morphologies and
behaviour. Research in cancer biology
indicates that several cancers are supported by
a small subset of cells with stem cell like
properties and are termed as cancer stem cells
(CSCs) or tumour initiating cells (TIC).
Evidences of CSCs involved in resistance to
conventional therapies, leading to metastasis
and tumour recurrence is abundant (Beck and
Blanpain, 2013; Chandler and Lagasse, 2010;
Prince and Ailles, 2008).
As early as 1937, Furth and colleagues
demonstrated that a single cell was able to
produce a haematopoietic malignancy on
implantation in mice (Furth et al., 1937). This
suggested that certain cells within a tumour
may have the ability to give rise to tumour
growth (Furth et al., 1937). Later, in 1994,
John Dick's group identified human acute
myeloid leukaemia-initiating cells using + -CD34 CD38 markers and showed that these
cells initiated tumour (Lapidot et al., 1994). In
1997, Bonnet and Dick showed for the first + -time that the CD34 CD38 population of cells
had the self-renewal property. The authors
performed limiting dilution assay to show that + -low numbers of CD34 CD38 cells were able
to form tumours in NOD/SCID mice, identical
to donors; whereas considerably higher
Key words: Cancer stem cells (CSCs); EMT, Epithelial to mesenchymal transition; Lineage tracing; β-catenin; NICD, Notch intracellular domain.*Corresponding Author: Sanjeev K. Waghmare, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.Email: [email protected]
Developmental Signalling in Maintenance and Regulation
of Cancer Stem Cells
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar,
Navi Mumbai, India
Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare*
Tissue stem cells self-renew throughout the life of an organism thereby maintaining tissue homeostasis and
prevent cancer. The major signalling pathways such as Wnt, Notch and Sonic hedgehog control the stem
cell regulation and their deregulation leads to cancer. Recent evidences showed that there exists a subset of
cells within tumour termed as cancer stem cells (CSCs). These CSCs escape the conventional chemo-
radiotherapy and further lead to tumour relapse followed by metastasis. This review focuses on the
developmental signalling pathways that are involved in the regulation and maintenance of normal stem cells
and CSCs. Understanding the molecular mechanism may be useful to specifically target the CSCs while
sparing the normal stem cells to reduce tumorigenecity.
Biomed Res J 2015;2(1):37-56
Review
+ +numbers of non-CSCs (CD34 CD38 ) were
unable to form tumours (Bonnet and Dick,
1997). These cells were coined as cancer stem
cells. In 2003, Michael Clarke's group
reported the first isolation of CSCs from breast
tumour (Al Hajj et al., 2003). Subsequently,
the presence of CSCs in other solid tumours
like melanomas, hepatocellular carcinoma,
glioblastoma, pancreatic cancer, colorectal
cancer and head and neck cancer have been
identified (Keshet et al., 2008; Li et al., 2007;
Ma et al., 2007; Prince et al., 2007; Ricci-
Vitiani et al., 2007; Singh et al., 2004). The
CSC markers from various cancers are listed in
the Table 1. The characterisation of CSCs uses
various assays that include: sphere-forming
assay, serial transplantation assay in NOD/
SCID mice and in vivo lineage tracing. Serial
transplantation assay, is considered as 'gold
standard' assay, and measures self-renewal as
well as the tumorigenic property of CSCs in
vivo (Al Hajj et al., 2003; Beck and Blanpain,
2013; Bonnet, 1997; Prince et al., 2007).
Recently the strongest evidence for existence
of CSCs has come from the lineage tracing
experiments in mice model for various cancers
such as glioblastoma, skin and colon cancers.
The assay showed that the individual
fluorescent tagged cells have the capability to
give rise to a tumour (Chen et al., 2012;
Driessens et al., 2012; Schepers et al., 2012).
Although many different markers for
CSCs have been identified in tumours of
different tissues, cells isolated by using these
markers are not a pure CSC population. Hence,
one of the major challenges is the isolation of a
pure population of CSCs. Recent study on
quantitative proliferation dynamics of hair
follicle stem cells showed the isolation of stem
cells based on their cell division. This suggests
that it may be possible to isolate pure stem cell
population (Waghmare and Tumbar, 2013;
Waghmare et al., 2008). Another challenge is
to understand how these CSC populations are
regulated and maintained. Therefore, it is
important to study the various signalling
pathways that are crucial for survival of CSC
population.
Biomed Res J 2015;2(1):37-56
38 Developmental signalling in cancer cells
Table 1: Cancer stem cell markers in various cancers
Cancer Cancer stem cell markers
Leukaemia CD34+CD38
- (Bonnet, 1997)
Breast Cancer CD44+CD24- (Hajj et al., 2003); ALDH1+ (Ginesteir et al., 2007); CD133+ (Wright et al., 2008)
Head and Neck
Cancer
CD44+ Lin
- (Prince et al., 2007); A1DH1+ (Clay et al., 2010; Krishnamurthy et al., 2010); CD133
+
(Zhang et al., 2010); CD10+ (Fukusumi et al., 2014); CD98+ (Matens de Kemp et al., 2013)
Pancreatic Cancer CD44+CD24-ESA+ (Li et al., 2007); c-Met (Li et al., 2011)
Liver Cancer CD133+ (Ma et al., 2007); CD90+ (Yang et al., 2008); CD13+ (Haraguchi et al., 2010); OV6+ (Yang et al.,
2008)
Glioblastoma CD133+ (Singh et al., 2004); SSEA1+ (Son et al., 2009), MET (De Bacco et al., 2009)
Melanoma ABCB5+ (Keshet et al., 2008)
Colorectal Cancer CD133+ (Ricci-Vitiani et al., 2007); CD166+ (Dalerba et al., 2007; Vermeulen et al., 2008); Lgr5+ (Barker
et al., 2007; Vermeulen et al., 2008), CD44+ (Haraguchi et al., 2008), CD44v6+ (Todaro et al., 2014)
Embryonic developmental process and
cancer stem cells
Development of an organism is regulated at
the molecular level by various signalling
pathways, and deregulation in these molecular
mechanisms leads to cancer formation. Recent
studies have shown various similarities
between cancer and development. During the
normal developmental process, undifferentia-
ted embryonic stem cells further differentiate
and give rise to the differentiated tissues of an
organism. Similarly in cancer, undifferentia-
ted CSCs are involved in tumour progression
that leads to metastasis (Bellacosa, 2013).
The embryonic stem cells have a core
transcriptional network comprising of
transcription factors like Oct4, Sox2 and
Nanog that contribute to self-renewal and
pluripotency (Boyer et al., 2005). Similarly,
lung CSCs showed elevated levels of Oct4 and
Nanog transcription factors (Chiou et al.,
2010). In head and neck cancer, CD44 variant
CD44v3 was shown to interact with Oct4-
Sox2-Nanog leading to CSC like properties
such as self-renewal and cisplatin resistance
(Bourguignon et al., 2012). Recently, it was
shown that the lineage ablation of Sox2-
expressing cells in both benign and malignant
skin squamous cell carcinomas resulted in
tumour regression indicating an important role
of Sox2 in tumour initiation and CSC
functions. Moreover, chromatin immuno-
precipitation analysis identified Sox2 target
genes involved in controlling tumour stemness
(Boumahdi et al., 2014).
Another important phenomena common
to both the CSCs as wells as the embryonic
stem cells is the occurrence of epithelial to
mesenchymal transition (EMT). During EMT,
the cells lose their polarity and acquire
migration capabilities that results in loss of
epithelial marker E-cadherin and simulta-
neous increase in mesenchymal marker N-
cadherin. During embryogenesis, EMT is
associated with gastrulation required for the
formation of the three germ layers. In cancer,
EMT leads to invasion, metastasis and cancer
stem cell-like phenotype (Kalluri, 2009;
Singh, 2010). A recent study showed that
Twist1, an EMT promoter protein, is expressed
during early stages of tumorigenesis and is
required for the initiation of skin tumours
(Beck et al., 2015).
All these indicate that regulation of
embryonic stem cells and CSCs share similar
mechanisms. Therefore, it suggests that
deregulation of various developmental
pathways are involved in cancer formation and
CSC regulation and maintenance. Hence,
studying the developmental signalling
pathways will shed light on the regulation of
CSCs.
Developmental signalling pathways and
CSCs
The various pathways which are deregulated
in cancer include Wnt, Notch, Hedgehog,
EGFR, PI3K, NFκB, etc. Among these, three
Dash et al. 39
Biomed Res J 2015;2(1):37-56
well-known pathways such as Wnt, Notch and
Hedgehog play an important role in the
development and normal homeostasis.
Conversely, deregulation of these pathways is
shown in CSC regulation and maintenance
(Ailles, 2012; Purow, 2012).
Wnt Pathway
Wnt pathway is evolutionarily conserved and
is involved in various organisms. It was first
discovered in Drosophila, when a mutation in
wingless (wg) gene led to a distinct phenotype
including absence of wings and halters. Later,
Nusse's group showed that the insertion of
Mouse Mammary Tumour Virus (MMTV) in
mice led to mammary tumour by proviral
activation of the int oncogene. The int
oncogene was later demonstrated as the mouse
homologue of the Drosophila wg gene. From
these two studies, a new nomenclature Wnt
(combination of wg and int) was obtained
(Nusse et al., 1984; Rijswijk et al., 1987;
Sharma, 2013).
There are 19 highly conserved Wnt
ligands discovered till date. These ligands are
secreted hydrophobic glycoproteins found to
be associated with cell membranes and extra-
cellular matrix. In Wnt producing cells, the
endoplasmic reticulum produces Wnt ligands,
lipid modified by porcupine (Mikels, 2006;
Willert et al., 2003). Wnt ligands can act
through two general categories of pathways:
canonical and non-canonical. The canonical
pathway is β-catenin dependent, while the 2+non-canonical pathways include Wnt/Ca and
Wnt/JNK pathways. In the canonical pathway
shown in Fig. 1, Wnt ligands bind to the
conserved cysteine rich domain (CRD) of the
frizzled receptors (Fz) which in turn forms co-
receptors complexes with low-density
lipoprotein like receptors (Lrp5/6). Further,
this interaction recruits the Dishevelled (Dsh)
Figure 1: Wnt Pathway. A) In the absence of the Wnt ligand, β-catenin is phosphorylated by destruction complex (APC,
CK1α, GSK3 and Axin) and is subjected to proteasomal degradation resulting in no transcription of the Wnt target
genes.B) In the presence of the Wnt ligand, the destruction complex is disrupted and thereby β-catenin enters the
nucleus and brings about the transcription of Wnt target genes. APC: Adenomatous Polyposis Coli; CK1α: Casein kinase
1α; GSK3: Glycogen synthase kinase 3; TCF: T cell factor; LEF: Lymphoid enhancing factor; Dsh: Dishevelled; LRP:
Low-density lipoprotein like receptors.
40 Developmental signalling in cancer cells
Biomed Res J 2015;2(1):37-56
protein to the cytoplasmic tail of Fz receptor
and brings about inhibition of destruction
complex surrounding β-catenin. The
components of the destruction complex
comprise of scaffold protein Axin, Glycogen
synthase kinase 3β (GSK3β), Casein kinase 1α
(CK1α) and adenomatous polyposis coli
(APC). In the absence of the Wnt ligands, the
destruction complex hyper-phosphorylates β-
catenin and targets it for proteasomal
degradation by ubiquitination. The binding of
Wnt ligand to Lrp5/6 causes phosphorylation
of the cytoplasmic tail of Lrp6, which in turn
recruits Axin to the receptor complex that
disrupts the destruction complex and stabilises
β-catenin. The stable β-catenin translocates to
the nucleus and binds to the lymphoid
enhancing factor/T-cell factor (LEF/TCF)
thereby transcriptionally activating the
different target genes involved in cell fate
determination during embryonic development
and tissue homeostasis (Mikels, 2006; Willert
et al., 2003).
Wnt signalling in normal development and
cancer
Wnt pathway is involved in different biological
processes such as embryonic development,
self-renewal, proliferation, morphogenesis,
etc. Wnt3a and Wnt1 knock out in mice led to
deficiencies in neural crest derivatives and
neural tube formation during the development
(McMahon et al., 1990; Yoshikawa et al.,
1997). Wnt3 knock out in mice led to early
gastrulation defect and perturbations in the
establishment of apical ectodermal ridge
during development (Liu et al., 1999). Further,
absence of Wnt4 ligand led to defects in female
development, while Wnt7a deletion led to
female infertility in mice (Jeays-Ward et al.,
2004; Parr et al., 1998). Axin1 knockout in
mice led to neuro-ectodermal and cardiac
abnormalities (Zeng et al., 1997). Wnt
signalling was shown to be crucial in hair
follicle development as targeted deletion of β-
catenin in the epidermis led to failure in
placode morphogenesis (Huelsken et al.,
2000). Absence of Lef1 led to defects in the
pro-B-cell proliferation and abnormalities in
several organs like teeth, mammary glands,
whiskers and hair (Reya et al., 2000;
VanGenderen et al., 1994); while the knockout
of Tcf1 led to thymocyte proliferation and
differentiation defects (Schilham et al., 1998).
Using the Wnt reporter, Axin2-LacZ, Wnt
responsive cells were localised to the sub
ventricular zone (SVZ) of the developing brain
and basal layer of the mammary ducts, which
are the stem cells niches. Furthermore, these
Wnt responsive cells showed high sphere
forming ability and were able to differentiate.
Hence, the Wnt pathway plays an important
role in normal development and tissue
homeostasis (Logan, 2004; VanAmerongen et
al., 2009).
There are strong evidences showing
involvement of Wnt pathway in regulation of
various cancers. Frequent somatic mutations
Biomed Res J 2015;2(1):37-56
Dash et al. 41
in β-catenin were observed in both mice and
human hepatocarcinomas (Coste et al., 1998),
prostate cancers and colon cancers (Voeller et
al., 1998). During intestinal adenoma
initiation, the first step was APC inactivation
followed by β-catenin stabilization, while
progression from adenoma to carcinoma
required the synergistic action of k-ras
activation and β-catenin nuclear localization
(Phelps et al., 2009). β-catenin was shown to
be essential for retaining tumorigenecity of
MDA-MB-231 breast cancer cell lines both in
vivo and in vitro. Further, β-catenin
knockdown cells implanted into mice showed
decrease in the tumour size. In addition, an in
vitro study in breast cancer cell lines showed
reduction in aldehyde dehydrogenase 1
(ALDH1) positive cells (Xu et al., 2015).
Wnt3a expression was associated with EMT
and promoted colon cancer progression (Qi et
al., 2014). Moreover, deletion of Axin1 was
reported in sporadic medulloblastomas and
hepatocellular carcinomas (Dahmen et al.,
2001). Increased expression of Dsh protein in
non-small cell lung carcinoma and meso-
thelioma have been reported (Uematsu et al.,
2003).
Wnt signalling in normal and cancer stem
cell regulation and maintenance
Wnt signalling is important in adult stem
regulation and has been shown to be involved
in stem cell proliferation, self-renewal and
maintenance. In hemato-poietic stem cells
(HSC), overexpression of β-catenin increases
the stem cell pool size suggesting that Wnt
pathway is critical to maintain the
hematopoietic stem cell homeostasis (Reya et
al., 2003). In mice hair follicle stem cells, live
cell imaging showed that β-catenin activation
in hair follicle stem cells was involved in hair
follicle tissue growth (Deschene et al., 2014).
Further, Wnt target gene Lgr5, a G-protein
coupled receptor was identified as an intestinal
stem cell marker indicating an important role
of Wnt pathway in the regulation of intestinal
stem cells (Ailles, 2012, Valkenburg, 2011).
The deletion of Tcf4, a Wnt downstream gene
showed loss of stem cell activity and reduced
proliferation of the intestinal epithelium
(Korinek et al., 1998). In addition, Lgr5 was
identified as a marker of hair follicle stem cells
(Jaks et al., 2008) with multipotent properties.
Moreover, Wnt inhibitor SFRP1 was shown to
play an important role in hematopoietic stem
cell maintenance through extrinsic regulation
(Renstrom et al., 2009). Over-expression of
Sfrp1 led to enhanced mesenchymal stem cell
function in angiogenesis (Dufourcq et al.,
2008). Besides, Sfrp1 was over-expressed in
hair follicle stem cells as compared to the non-
stem cells (Tumbar et al., 2004; Zhang et al.,
2009). Recently, it was shown that Sfrp1 gene
is critical for maintaining proper mammary
gland development wherein loss of Sfrp1
promotes mammosphere formation; however
the role in mammary stem cells needs further
investigation (Gauger et al., 2012).
42 Developmental signalling in cancer cells
Biomed Res J 2015;2(1):37-56
In cancer, various reports have shown that
deregulation of Wnt pathway is crucial for the
CSC regulation. Human head and neck CSCs
treated with Wnt antagonist, secreted frizzled-
related protein 4 (sFRP4), the CSCs showed
reduction in the sphere-forming capacity and
decrease in the stemness markers like CD44
and ALDH1 (Warrier et al., 2014). In another
report, β-catenin was shown to be required for
maintenance of cutaneous CSCs since deletion
of β-catenin led to reduction in the CSCs and
tumour regression (Malanchi et al., 2008).
CSCs isolated from mammary tumours of
radiation treated p53-null mice showed altered
DNA repair in response to radiation as well as
β-catenin activation (Zhang et al., 2010). In
prostate cancer, Wnt signalling induced
tumour initiation, EMT and metastasis.
Additionally, in prostate cancer cell lines and
primary cultures, Wnt3a treatment increased
the self-renewal capacity of putative prostate
CSCs, emphasizing that Wnt signalling plays
an important role in prostate cancer (Barker,
2006; Valkenburg, 2011; Verras et al., 2004).
Moreover, the inactivation of APC in Lgr5-
positive stem cells at the intestinal crypts led to
transformation within days; while inactivation
of APC in progenitors or differentiated cells
did not lead to tumour formation even after 30
weeks (Barker et al., 2009). In addition, the
deletion of CD44, a CSC marker and a Wnt
target gene in mice having heterogeneous APC Min/+
mutation (APC ), attenuates intestinal
tumorigenesis (Zeilstra et al., 2008).
Notch Pathway
Notch gene was first discovered in Drosophila
by Morgan and Bridges where they showed
that a mutation led to wings notching and
hence the name “Notch” was coined (Morgan
and Bridges, 1916; Mohr, 1919; Poulson,
1940). There are four Notch genes, three
Delta-like and two Jagged genes in mammals,
that are translated into different Notch ligands,
Delta and Jagged. Recently, it was shown that
for cell fate determination during
development, complex of Notch receptor-
Delta-Jagged acts in concert (Fiuza, 2004;
Boaretoa et al., 2015).
Since the Notch ligands such as Delta and
Jagged proteins, as well as Notch receptors are
transmembrane proteins, cell-cell contact is
important for the signalling cascade. The
Notch receptors contain an extracellular
subunit, having multiple EGF-like repeats,
and a transmembrane subunit (Wharton et al.,
1985). When the Notch ligand binds to its
receptor, the extracellular domain of the Notch
receptor is dissociated from the trans-
membrane domain and the S2 cleavage site is
exposed (Fig. 2). This site is cleaved by
ADAM (a disintegrin and metalloprotease)
generating an intermediate that is further
cleaved by γ-secretase to generate Notch
Intracellular Domain (NICD). NICD then
translocates to the nucleus where it binds to
ubiquitous transcription factor CSL (CBF-1,
Suppressor of Hairless, Lag-1). This complex
displaces a co-repressor complex containing
Biomed Res J 2015;2(1):37-56
Dash et al. 43
SKIP, SHARP and histone deacetylases.
Further, it recruits a co-activator complex
containing a MAML (Mastermind-like
protein), p300 and other chromatin modifying
enzymes, thereby bringing about transcription
of different Notch target genes (Ailles, 2012;
Andersson, 2011; Fiuza, 2004).
Notch signalling in normal development
and cancer
Notch pathway is also evolutionarily
conserved and is important in cell to cell
communication that regulates cell fate
determination during development, cell
proliferation, differentiation and apoptosis.
The loss of Notch function in vertebrates is
associated with disruption of neurogenesis,
somite formation, angiogenesis, and lymphoid
development. In Drosophila, Notch is shown
to control the fate of various cell types in the
eye. In vertebrates, Notch is involved in the
establishment of the central and peripheral
nervous systems, spermatogenesis, oogenesis,
myogenesis and imaginal disc development
(Artavanis-Tsakonas et al., 1999). In normal
mammary development, Notch pathway
activation is required for regulation of cell
fate, proliferation and stem cell self-renewal.
The Notch pathway is also shown to be
important for tip-cell formation during
mammalian astrocyte differentiation and
angiogenesis. In vertebrates, the Notch
pathway leads to patterning during inner ear
hair cell formation and insulin-secreting
pancreatic β cell production (Ailles, 2012;
Fortini, 2009).
Notch signalling has been shown to be
involved in various cancers. For instance,
Notch1 regulates breast cancer cells by
inducing Slug expression (Shao et al., 2015).
Notch4 promotes growth of gastric cancer
cells through activation of Wnt1, β-catenin
Figure 2: Notch Pathway. A) In the absence of the Notch ligands (Delta and Jagged), the S2 cleavage site remains
hidden and inaccessible to ADAM. Hence, the NICD is not formed, with consequent no transcription of the Notch target
genes. B) In the presence of the Notch ligands, the conformational change in the intracellular subunit of the Notch
receptor takes place exposing the S2 cleavage site, thus leading to the formation of NICD. Further, NICD translocates the
nucleus and brings about transcription of Notch target genes. NICD: Notch intracellular domain; ADAM: A disintegrin and
metalloprotease; MAML: Mastermind-like protein; SKIP: Ski-interacting protein; SHARP: SMRT associated protein.
44 Developmental signalling in cancer cells
Biomed Res J 2015;2(1):37-56
(Quian et al., 2015) and the downstream target
such as c-myc and cyclin-D1. In mice, Notch1
activation combined with p53 loss showed
synergistic effect in the formation of
Osteogenic sarcoma (Tao et al., 2014).
Notch signalling in normal and cancer stem
cell regulation and maintenance
Notch has been shown to be involved in self-
renewal, proliferation and differentiation of
adult stem cells in various tissues. In mice
mammary stem cells, the knockdown of Cbf-1,
a canonical Notch effector, showed increased
stem cell activity in vivo suggesting a role in
controlling mammary stem cells. (Bouras et
al., 2008). Notch directly targets the crypt base
columnar cells that maintain stem cell activity
in mice (VanDussen et al., 2012). The Notch
activation maintains the slow cycling property
of neural stem cells; however, blocking Notch
resulted in increased number of stem cell
divisions followed by depletion of the stem
cell pool (Chapouton et al., 2010).
Furthermore, constitutive activation of Notch
signalling promotes self-renewal in muscle
stem cells through upregulation of Pax7 (Wen
et al., 2012). Mice with satellite cell specific
deletion of RBP-Jj (recombining binding
protein-Jj), a nuclear factor required for Notch
signalling, showed depletion of the stem cell
pool and their muscles lacked ability to
regenerate in response to injury (Bjornson et
al., 2012). Notch 3 has been shown to be
expressed in stem cells and disruption led to
defective stem cells proliferation (Kitamoto
and Hanaoka, 2010).
Notch signalling plays an important role in
a number of hematopoietic and solid tumours,
but the strongest evidences for its role in CSC
regulation has been shown in breast cancer,
embryonal brain tumours and gliomas (Fan et
al., 2006; Pannuti et al., 2010). In various
human breast cancer cell lines and primary
patient tissues, a significant decrease in
mammosphere formation after Notch
inhibition has been demonstrated (Abel et al.,
2014). Further, studies on the human
mammary mammospheres have shown a
feedback loop between Her2/Neu and Notch,
as well as promotion of a hypoxia resistant
phenotype (Pannuti et al., 2010). In brain
tumours, blockade of Notch led to a 5-fold +reduction in the CD133 cell fraction and total
depletion of the side population cells.
Additionally, differentiated cell growth was
observed after Notch inhibition, but lacked
formation of tumour xenografts efficiently,
indicating that the CSCs required for tumour
propagation were absent (Fan et al., 2006). A
recent study on primary human pancreatic
xenografts showed upregulation of the notch
pathway components in pancreatic CSCs.
Additionally, inhibition of notch pathway
reduced CSC percentage and tumour-sphere
formation significantly (Abel et al., 2014).
Biomed Res J 2015;2(1):37-56
Dash et al. 45
Sonic-hedgehog Pathway
Hedgehog pathway is delineated in
Drosophila and determines the anterior-
posterior orientation of developing structures
(Nusslein-Volhard and Wieschaus, 1980).
Similar to Wnt and Notch, the key components
of the Hedgehog pathway are evolutionarily
conserved, although differences are observed
in the mammalian and Drosophila Hedgehog
signalling. While Drosophila has only one
hedgehog gene, three homologues have been
identified in vertebrates namely, Sonic (Shh),
Desert (Dhh), and Indian hedgehog (Ihh). The
Sonic Hedgehog pathway is extensively
investigated in the vertebrate system (Chen et
al., 2005; Varjosalo et al., 2006). The other
components of the Hedgehog pathway include
patched (Ptc) and smoothened (Smo),
constituting a 12-pass transmembrane
glycoprotein and a 7-pass transmembrane
protein, respectively (Varjosalo, 2008;
Wicking et al., 1999).
In the Sonic-hedgehog pathway
elaborated in Fig. 3, absence of the hedgehog
ligand, Smoothened (Smo) is inhibited by
being bound to Patched (Ptc). When the
hedgehog ligand binds to Ptc, inhibition of
Smo is released which acts on protein complex
comprising of fused (Fu), suppressor of fused
(Sufu) and cos-2-costa-2 (Wicking et al.,
1999; Merchant, 2010). These proteins are
generally bound with Gli thereby inhibiting its
action. Once the complex is disrupted, Gli
translocates to the nucleus and brings about
transcription of different downstream targets
(Sasaki et al., 1999; Ruiz, 2007; Stecca, 2010).
Sonic-hedgehog signalling in normal
development and cancer
In vertebrates, the Sonic Hedgehog (Shh), is
expressed widely throughout the developing
central nervous system (CNS), limb, gut, teeth
and hair-follicle. Dhh is involved in
development of the germline, while the Ihh is
Figure 3: Hedgehog Pathway. A) In the absence of Hedgehog ligands (Indian, Sonic and Dessert), the Patched
receptor (ptc) exerts inhibitory action on the Smoothened receptor (smo). The Gli complex (Gli1 and Gli2) remains in the
cytoplasm followed by no transcription of Hedgehog target genes. B) In the presence of Hedgehog ligands, the inhibitory
action of Patched (ptc) on Smoothened (smo) is released, and hence Gli complex translocates to the nucleus and brings
about transcription of Hedgehog target genes.Fu: Fused; SuFu: Suppressor of Fused; Cos 2: cos-2costa-2.
46 Developmental signalling in cancer cells
Biomed Res J 2015;2(1):37-56
involved in development of the skeletal system
(Bitgood et al., 1996; Wicking et al., 1999).
Shh also plays a role in neural stem cells,
determining the neuronal cell fate (Merkle et
al., 2007). It was demonstrated that during
repair of acute airway injury, the Hedgehog
pathway gets activated in the airway
epithelium (Watkins et al., 2003). Hedgehog
signalling components Ptc, Gli1, and Gli2
were over-expressed when mammary
progenitor cells grow as mammospheres (Liu
et al., 2006). These reports indicate that the
Hedgehog pathway plays a role in normal stem
cell regulation (Ailles, 2012).
Hedgehog signalling is involved in
various cancers. For example, Ptc1 mutation
was observed in patients with medullo-
blastoma and rhabdomyosarcoma (Hahn et al.,
1996; Johnson et al., 1996; Pietsch et al.,
1997). Sufu as well as Smo mutations were
observed in medulloblastoma (Xie et al., 1998;
Taylor et al., 2002); Gli1 and Gli3 mutations
were seen in pancreatic adenocarcinoma; and
Gli1 gene amplification was seen in
glioblastoma (Clement et al., 2007; Jones et
al., 2008). Further, Kern et al showed that Gli
and PI3K/AKT/mTOR signalling act
synergistically to initiate and maintain chronic
lymphocytic leukemia (Kern et al., 2015).
Another report showed that Sonic hedgehog
ligand over-expression led to increased
number and size of intestinal adenomas in
APC (HET) mice, while loss of Indian
Hedgehog almost completely blocks intestinal
adenoma development (Buller et al., 2015).
Sonic-hedgehog signalling in normal and
cancer stem cell regulation and
maintenance
Hedgehog signalling is involved in stem cell
regulation of various tissues. Shh regulates
self-renewal of neural stem cells (Palma et al.,
2005). The components of Hedgehog pathway
such as Ptc, Gli1 and Gli2 are expressed in the
mammary stem cells and down regulated
during differentiation (Liu et al., 2006).
Hedgehog is involved in controlling neural
stem cells through the p53-independent
regulation of Nanog (Po et al., 2005).
In colon carcinoma, Hedgehog signalling
is activated in CSCs with higher expression of
Gli1 and Gli2. In non-small cell lung cancer,
the malignant phenotype of the tumours is
maintained by ligand-dependent Hedgehog
pathway activation (Watkins et al., 2003).
Furthermore, Bmi1, which is a downstream
target of the Hedgehog pathway was activated
in breast CSCs and is also shown to regulate
normal and leukemic stem cells (Liu et al.,
2006; Takebe, 2011).
CONCLUSION
Tumour maintenance and progression is
regulated by a subset of cells that are known as
cancer stem cells (CSCs). Recently, due to
increase in evidences on the existence of
Biomed Res J 2015;2(1):37-56
Dash et al. 47
CSCs, they have gained more attention but
how these CSCs escape the chemo-
radiotherapy is still unknown. Moreover, how
the CSCs are maintained in the tumour micro-
environment remains elusive. Several reports
showed signalling pathways such as Wnt,
Notch and Sonic-hedgehog are deregulated in
cancer, and also involved in the CSC
regulation and maintenance. In addition,
evidence of cross-talk between the signalling
pathways exists. Therefore, understanding
these signalling pathways at the molecular
level will be of utmost importance The study .
will enable counteracting the issue of
signalling cross-talk, and perhaps, multi
targeted drugs approach can be fruitful.
Hence, further detailed research on
deregulation of the developmental pathways
in CSCs needs to be investigated. Eventually,
elucidation of the signalling mechanisms will
enable to specifically target CSCs without
affecting the normal cells.
ACKNOWLEDGEMENTS
The authors acknowledge Mr. Rahul Sarate
and Mr. Gopal Chovatiya for their
suggestions.
CONFLICT OF INTEREST
The authors claim no conflict of interest.
Biomed Res J 2015;2(1):37-56
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