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: swaghmare@actrec.gov.in

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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