Accepted Manuscript

Mini-review

Inflammation and cancer stem cells

Sarah Shigdar, Yong Li, Santanu Bhattacharya, Michael O’Connor, Chunwen

Pu, Jia Lin, Tao Wang, Dongxi Xiang, Lingxue Kong, Ming Q. Wei, Yimin Zhu,

Shufeng Zhou, Wei Duan

PII: S0304-3835(13)00551-X

DOI: http://dx.doi.org/10.1016/j.canlet.2013.07.031

Reference: CAN 11572

To appear in: Cancer Letters

Please cite this article as: S. Shigdar, Y. Li, S. Bhattacharya, M. O’Connor, C. Pu, J. Lin, T. Wang, D. Xiang, L.

Kong, M.Q. Wei, Y. Zhu, S. Zhou, W. Duan, Inflammation and cancer stem cells, Cancer Letters (2013), doi: http://

dx.doi.org/10.1016/j.canlet.2013.07.031

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Inflammation and cancer stem cells

Sarah Shigdara*, Yong Lib, Santanu Bhattacharyac, Michael O’Connora, Chunwen Pud, Jia

Lina, Tao Wanga, Dongxi Xianga, Lingxue Konge, Ming Q. Weif, Yimin Zhug, Shufeng

Zhouh, and Wei Duana*

a. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria, 3217,

Australia

b. Cancer Care Centre, St. George Hospital, and St George Clinical School, Faculty of

Medicine, University of New South Wales, Kensington, NSW 2052, Australia

c. Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012,

India

d. Dalian Sixth People's Hospital, Dalian, China，116033

e. School of Medical Science and Griffith Health Institute, Griffith University, Gold

Coast Campus, Southport, Australia

f. Institute for Frontier Materials, Deakin University, Waurn Ponds, Victoria, Australia

g. Suzhou Key Laboratory of Nanobiomedicine, Division of Nanobiomedicine, Suzhou

Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou,

Jiangsu, China

h. Department of Pharmaceutical Sciences, College of Pharmacy, University of South

Florida, Tampa, Florida, U.S.A.

*Corresponding authors: sarah.shigdar@deakin.edu.au and wduan@deakin.edu.au

Key words, Cancer stem cells, inflammation, microenvironment, epithelial-mesenchymal transition, epigenetics

Abstract

Cancer stem cells are becoming recognised as being responsible for metastasis and treatment

resistance. The complex cellular and molecular network that regulates cancer stem cells and

the role that inflammation plays in cancer progression are slowly being elucidated. Cytokines,

secreted by tumour associated immune cells, activate the necessary pathways required by

cancer stem cells to facilitate cancer stem cells progressing through the epithelial-

mesenchymal transition and migrating to distant sites. Once in situ, these cancer stem cells

can secrete their own attractants, thus providing an environment whereby these cells can

continue to propagate the tumour in a secondary niche.

1. Introduction

Cancer is still one of the most devastating diseases in the world with only a modest

improvement in 5-year survival rates seen with ‘new and improved’ cancer therapies [1]. The

high complexity and diversity seen in cancer development, and the intricate relationship with

the micro-environment is still being unravelled. The last two decades have provided more

knowledge regarding cancer than possibly the previous 50 years, and the traditional cancer

model has been re-written several times in that period. One of the most enlightening

discoveries has been the identification of a population of cells that is thought to be

responsible for chemo-resistance and relapse after treatment [2, 3]. As these highly

tumourigenic cancer cells display some functional properties of normal stem cells, such as

self-renewal, multi-potent differentiation, and have the capacity to generate a heterogeneous

lineage of all types of cancer cells comprising a tumour, they are often referred to as cancer

stem cells (CSCs) [4-7]. Based on the CSC hypothesis, the bulk of cells in the tumour are

composed of non-CSCs which are non-tumourigenic and have limited detoxification enzymes

and potential for proliferation [3]. In contrast, a relatively small proportion of cells within a

tumour mass consist of CSCs, which have co-opted many of the features of normal stem

cells, and have a slower proliferation rate, a protective mechanism for DNA repair and

upregulated expression of anti-apoptotic genes [8, 9], as well as resistance to drugs and toxins

via the expression of several ATP-binding cassette (ABC) transporters. Indeed, CSCs are

thought to be in a relatively quiescent state, therefore evading chemotherapeutic challenges,

and thus ‘protecting’ the continuity of the tumour [10], as well as potentially endowing the

ability to propagate and sustain tumourigenesis in a cancer [11-14].

The idea that there are subpopulations within the tumour that may promote tumour formation

and be more relevant for predicting a therapeutic response has gathered steam over the last

few years. However, what is still relatively unknown is how these cells develop and just

exactly ‘when’ they originate. It still remains to be established whether the CSC is a function

of a specific cell type, the micro-environment, epigenetic and genetic changes, or the stage of

the tumour [15]. As well, there might be other cues received from other cells, such as

cytokines, that may promote the formation of this population of cells. Given that a link has

been established between chronic inflammation and the development of cancer, this review

will investigate the roles that signalling pathways, cytokines, growth factors and epigenetics

play in linking inflammation and the CSC phenotype.

2. Cancer and Inflammation

Solid tumours trigger an inflammatory response during cancer progression. This

inflammatory response modifies the tumour microenvironment, with an influx of immune

cells – macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, dendritic

cells, natural killer cells and T and B lymphocytes. This influx adds to the heterogeneous

mixture of cells already surrounding the tumour in the stroma – the fibroblasts, endothelial

cells, pericytes and mesenchymal cells. Communication occurs via chemokines and cytokines

or through direct contact. Whether the immune reaction is pro or anti-tumour depends on

which cell type is in abundance and the status of activation [16]. It is this process that

limits the number of tumours that are diagnosed as the immune system is very effective in

removing the majority of tumours [17]. The tumour does not play a passive role in this

process, with tumour cells also producing cytokines and chemokines to attract immune cells

[18]. Once the tumour is established, additional environmental cues cause the cancer cells to

metastasise through a process known as the epithelial-mesenchymal transition (EMT) [19].

3. Cancer formation, EMT and CSCs

To investigate the role that inflammation has on CSCs, we first need to understand their

origins. One of the issues with the term cancer stem cells and cancer initiating cells is that

these monikers are used interchangeably. These terms are derived from the ability of a certain

population of cells being able to propagate a tumour in vivo upon transplantation into an

animal model. However, these terms are perhaps misnomers, as these cancer stem cells or

cancer initiating cells are not necessarily the cell of origin of the initial primary tumour [6].

An initial oncogenic hit to a cell will eventually, over a long period of time and following the

accumulation of other oncogenic ‘hits’, lead to a cancerous tumour (Fig 1). The

accumulation of proliferating tumour cells takes on the resemblance of chronic inflammation

and the activated stroma produces a number of cytokines, angiogenic factors and chemokines,

which in turn recruit additional immune cells, leading to a ‘ball of cells’ which resembles a

‘never healing wound’ [20]. The centre of this ‘ball’ becomes hypoxic, which leads to the

upregulation of hypoxia inducible factor-1α (HIF-1α), Snail and Twist1, as well as the

activation of Notch or NF-κB pathways and induction of DNA hypomethylation [21]. Thus

starts the process of the epithelial-mesenchymal transition (EMT) (Fig 2). This EMT is a

cellular program that confers the biological traits needed to accomplish the majority of the

steps of the invasion-metastasis cascade onto cancerous epithelial cells [22]. It has been

shown that cancer cells can de-differentiate into CSCs under certain circumstances – a

process termed plasticity [19, 23]. This could be the first step in the process for cancer cells

to become CSCs followed by progression through the EMT and migration to secondary sites.

The aberrant activation of EMT, and the subsequent dedifferentiation has been shown to

strongly enhance cancer cell motility and dissemination, with gene expression patterns

indicating these dedifferentiated cells combine EMT properties with a stem-cell like

phenotype [24]. Thus, the concept of migrating CSCs, promoted by chronic inflammation, as

the basis of metastasis was formed [25].

4. Pathway activation and transcription factors as components of CSC/EMT

Normal stem cells display plasticity and dedifferentiation in the right environmental setting or

under the influence of an experimental manipulation [26-29]. Similarly, cancer cells undergo

genotype and/or phenotype changes and convert to a CSC-state due to their immature

phenotype and derailed signal transduction, with a number of pathways havinge implicated in

CSC/EMT progression (Fig 2). The first step is for cancer cells to lose their adhesion to their

fellow cells and become motile. Cancer cells typically attach themselves to each other via

connected adherens junctions. This forms a rigid structure to the actin skeleton via E-

cadherin, α-, β-, and γ- catenins [30]. During the EMT process, these proteins are

downregulated, thus removing the tethering to adjacent cells and allowing them to move [30].

Transcription factors, such as Snail, Slug, Twist, Six1, Lbx1, Cripto, Zeb1, Zeb2, E12, E46,

oncostatin-M and NF-κB have been shown to act as EMT regulators and a number of

signalling pathways have been shown to be activated [31-38].

4.1. NF-κB Pathway

The NF-κB pathway is one of the most interesting links between inflammation, CSCs and

EMT. Several different stimuli have been shown to activate the NF-κB pathway. These

include the hypoxia inducible factor-1 alpha (HIF-1α) which is induced in normoxic and

cancer cells following stimulation by pro-inflammatory cytokines, such as tumour necrosis

factor alpha (TNF-α) or interleukin-1 beta (IL-1β) [18]. It is also activated by IL-6 and it is

the upregulation of the NF-κB pathway by IL-6 that is thought to play a role in trastuzumab

resistance in breast cancer [39]. Following the activation of the NF-κB pathway, several

downstream events occur, such as PTEN and apoptosis being suppressed. As well, the EMT

regulators, Snail and Slug, are activated which leads to an EMT phenotype. Following

activation of Snail or Slug, the adhesion protein, E-cadherin, is downregulated, thus leading

to separation of cancer cells [39], the first step in the EMT process. The anti-diabetes drug

metformin, has been shown to inhibit the nuclear translocation of NF-κB and phosphorylation

of Stat3 in CSCs [40]. However, inhibition of NF-κB in vivo has been shown to enhance the

growth of squamous cell carcinoma and promote the development of hepatocellular

carcinoma, while in mammary epithelial cells, NF-κB promoted initial onset and growth of

tumours, as well as the infiltration of tumour-associated macrophages. Subsequent inhibition

of NF-κB retarded EMT and breast CSC expansion [41], indicating the conflicting role that

NF-κB plays in tumour formation.

4.2. Wnt/β-catenin Pathway

The canonical Wnt/β-catenin pathway is very much dependent on the amount of β-catenin in

the cytoplasm [42]. Extracellular Wnt binds to Frizzled cell surface receptors and low-density

lipoprotein receptor-related protein (LRP) co-receptors, which activates Dishevelled. This, in

turn, mediates the proteolytic degradation of β-catenin, resulting in an increase of β-catenin

levels. The downregulation or loss of E-cadherin leads to β-catenin translocating to the

nucleus, where it induces the expression of target genes that promote motility and inhibit

adhesion [43, 44]. Wnt signalling has been shown to be crucial in maintaining stemness in

normal colon stem cells. Indeed, this pathway is quite commonly deregulated in colon

cancers with mutations leading to defective β-catenin degradation resulting in an

accumulation in the nucleus which results in the continuing transcription of Wnt target genes

[45]. A recent study has shown that cells with high Wnt pathway activity is observed in

cancer cells close to stromal myofibroblasts [45]. Hepatocyte growth factor (HGF), secreted

by the myofibroblasts, was able to activate β-catenin dependent transcription and thus CSC

clonogenicity. This interaction was also able to activate CSC plasticity, with a restored CSC

phenotype seen in more differentiated cancer cells following exposure to conditioned media

derived from myofibroblasts [45].

4.3. Notch Pathway

The Notch pathway is also well recognised as playing a role in CSCs. Notch binds to a Delta

or Jagged type ligand and is then cleaved with the intracellular portion being translocated to

the nucleus [37]. This process activates p21, a cell cycle inhibitor [46, 47]. Interestingly,

Notch is activated in cells in the immediate vicinity of active tumour progenitors, suggesting

that Notch drives a quiescent phenotype via some feedback control mechanism [48]. Studies

have shown that the population of CSCs is tightly controlled with xenograft studies proving

that a combined implantation minimises the effect of CSCs. Once Notch is activated and thus

activates p21, several other signalling cascades, such as Myc, signal transducer and activator

of transcription (Stat) 3, and Wnt4 are blocked [49]. Notch does have conflicting roles in

tumour progression however. While high levels have been observed in lung cancer stem cells

and high levels have been shown to induce quiescence in pancreatic endocrine progenitors,

lower levels have been shown to promote progenitor amplification [50, 51]. Notch signalling

has also been shown to be required for survival and proliferation in CSCs with a reduction in

CSC frequency and growth observed following Notch1 inhibition [52].

4.4. Sonic Hedgehog (Shh) Pathway

The Sonic Hedgehog (Shh) pathway generally acts as a tumour suppressor through the

actions of its negative regulator, Patched [53, 54]. It binds to the Patched transmembrane

receptor, which in turn inhibits the transmembrane protein, Smoothened, which via a negative

feedback mechanism, inhibits further activation of the Shh pathway [37]. Without Shh,

Patched inhibits Smoothened resulting in inactivation of the Shh pathway [55]. Uncontrolled

activation of Shh signalling though loss-of-function mutations in the negative regulators, such

as Patched, results in constitutively activation and overproduction of Shh in human cancers,

such as basal cell carcinoma, medulloblastoma, stomach, lung, pancreatic and oesophageal

carcinomas [56]. Studies have shown that the Shh pathway is activated by TNF-α and IL-1β

in pancreatic cells, suggesting that inflammatory stimuli enhanced proliferation [57]. In

addition, Shh signalling was shown to be stimulated by IL-6 in acute myeloid cells in vitro

[58]. When the Shh pathway is aberrantly activated in cancers, several downstream events

occur. These include the upregulation of several EMT regulators such as Nanog, Oct-4,

VEGF, PDGFR-α and Snail [59]. A link has been shown between the aberrant expression of

Shh and EMT through chemical inhibition of the Hedgehog pathway. Cyclopamine is a

steroidal jerveratrum alkaloid that has successfully been used to study the role of the Shh

pathway in normal and aberrant development. The use of this inhibitor reduced expression of

Snail and upregulated E-cadherin in pancreatic cell lines, changes associated with the

suppression of EMT [60]. This effect has also been seen in gastric cancer tumoursphere

models [55]. As well, the use of sulforaphane has been shown to downregulate downstream

targets of the Shh/Gli pathway, such as Nanog, Oct-4, VEGF and PDGFR-α, reduce the

expression of EMT markers which led to a marked reduction in EMT and reduced tumour

growth in a mouse model of pancreatic cancer [59]. Interestingly, recent reports have shown

that the NF-κB pathway regulates Shh expression and it is the upregulation of Shh by NF-κB

that promotes proliferation [61]. This relationship has been demonstrated in breast cancer,

with breast cancer cell lines showing Shh upregulation with a concomitant expression of NF-

κB [62]. Of note, a study by Yauch et al has shown that it is the non-malignant stromal cells

surrounding the tumour that respond to secreted proteins, thus activating the Shh pathway in

these cells to support tumour growth and proliferation [63].

4.5. p53

p53, in its classical form is a tumour suppressor and provides the primary genetic defence

against cancer by inducing DNA damage responses to various genotoxic stresses, such as

programmed cell death or cell-cycle arrest [64-66]. However, it is the most frequently

mutated tumour suppressor in human cancers [65]. Sustained activation of p53 has been

shown to lead to pro-tumourigenic inflammation in the liver, triggering high-mobility group

box 1 (HMGB1) release through late apoptosis, necrotic cell death and loss of membrane

integrity. This release of HMGB1 attracts leucocytes and induces these leucocytes and

resident immune cells to release TNF-α, IL-1, IL-6 and other cytokines [67]. Indeed, p53

plays a vital role in the inflammatory response as demonstrated in a recent study using a p53

knock-out rat [64]. In this study, the extent of inflammatory cytokines, such as IL-6 and TNF-

α, were much lower in p53+/- rats. This study showed that DNA damage activated p53 and

induced apoptosis, which triggered the release of inflammatory signals in the surrounding

microenvironment, thus promoting the transformation and clonal expansion of genetically

mutated cells in the liver [64]. In addition to hepatocellular carcinoma, similar results were

obtained in p53-deficient mice that developed a lower incidence of chemically induced skin

tumours, which also involves a large inflammatory component [64, 68]. These results do

appear to be contrary to the role that p53 plays in tumour development in other organs. For

example, in ovarian cancer cells with low or mutated p53 expression, a high level of

proinflammatory chemokines, such as CXCL, 2, 3, and 8, was observed. Furthermore,

transient expression of p53 into these deficient cells downregulated proinflammatory

chemokines induced by TNF-α. p53 restoration also blocked the TNF-α induced NF-κB

promoter activity [69]. Gene expression studies in p53 deficient colorectal cancer models

showed significant upregulation in genes associated with chemotaxis, prostaglandin synthesis

and inflammation, as well as genes associated with EMT, such as Twist, versican, tenascin C

and fibronextin 1 [66]. A number of the genes upregulated were well-established NF-κB

target genes. CXCL1 was strongly expressed along the invasive front of the tumour, a

chemokine associated with being a potent chemoattractant of myeloid cells. The authors of

this study suggest that p53 plays a suppressive role on the inflammatory microenvironment

associated with NF-κB during the initial progression stage. Once p53 is lost in the first few

cancer cells, the equilibrium between p53 and NF-κB is shifted. Once the epithelial barrier

function is impaired and NF-κB activation follows due to the leakage of intestinal bacteria ,

myeloid cells are recruited to the tumour microenvironment through the induction of

chemoattractants thus begins the process of EMT [66]. A further study has linked mutated

p53 with prolonged TNF-α-induced NF-κB activation in cultured colorectal cell lines and

non-epithelial cells such as embryonic fibroblasts, a result later confirmed in mice harbouring

a germline p53 mutation that were more prone to inflammatory-associated colorectal cancer

[70].

5. Linking Inflammation to CSCs

All of these pathways have been shown to be integral to CSCs and EMT. As well, given the

link of Notch to quiescence, it is likely that this is an important pathway linking inflammation

with CSCs. But how are these pathways activated, and is it a component of inflammation that

is responsible for activating the pathways responsible for the development of CSC and

progression through EMT? Well recognised mediators of these pathways are inflammatory

cytokines (Fig. 3). There are a number of inflammatory cytokines that are released during

chronic inflammation [71]. Transforming growth factor-beta (TGF-β) has the ability to

regulate immune cell populations and suppress the activity of T-cells, NK cells, neutrophils,

monocytes and macrophages involved in repressing tumour formation and progression with

the balance between suppression and promotion of tumour progression regulated by TGF-β in

vivo [72]. TGF-β activates Smad transcription factors which in turn activates Snail and Slug,

and suppresses E-cadherin, which are classical steps in EMT. Indeed, this phenomenon has

been observed in breast cancer where the binding of TGF-β resulted in an EMT phenotype

[73]. TGF-β also recruits various immune cells into the tumour microenvironment where the

resulting inflammation leads to the secretion of growth factors, cytokines, chemokines,

proteases and extracellular matrix modifying enzymes [72]. The tumour associated

macrophages (TAMs) play a vital role in the progression of cancer [74]. These TAMs secrete

a number of factors, including IL-1 and TNF-α which have been shown to support the steps

of invasion and metastasis [74]. TNF-α can upregulate TGF-β and accelerate TGF-β induced

EMT. TNF-α can also induce NF-κB and Snail directly and stabilises regulators of EMT [16].

IL-6 is secreted by inflammatory cells and cancer cells exposed to IL-6 possess malignant

properties, such as enhanced capacity for invasion and increased drug resistance [74, 75]. A

recent study using PTEN knockdown breast cancer cells showed that trastuzumab treatment

enriched for CSCs which displayed an EMT phenotype and secreted 100-fold more IL-6 than

the parental cells. Expression studies also showed IL-6 activated the Akt, Stat3 and NF-κB

pathways [39]. Once IL-6 binds to its receptor, the Jak/Stat3 pathway is activated, with IL-6

being a major activator of Stat3 in breast cancer cell lines. This forms a major positive

feedback loop, with Stat3 enhancing the production of IL-6. It is also thought that NF-κB is

involved in IL-6 mediated Stat3 activation through the repression of the microRNA Let-7, a

direct repressor of IL-6 [75]. The IL-6/Stat3 pathway has also been shown to be important in

converting non-CSCs into CSCs [76]. IL-6 is one of the cytokines that has been shown to be

essential for protecting CSCs from chemotherapy. IL-6 upregulates survival of breast cancer

cells by promoting Notch3/Jagged1 signalling pathway which promotes cell renewal, hypoxia

survival and invasiveness potential, classical characteristics of CSCs[77]. Interestingly, the

NF-κB and Stat3 pathways converge on the EMT, with IL-6 mediated Stat3 activation

inducing the E-cadherin repressor, Snail, with NF-κB promoting post-transcription

stabilisation of the Snail protein [20]. IL-6 has also been shown to attract and activate

mesenchymal stem cells, which then produce a CSC supporting chemokine, CXCL7 [78].

Another type of cell that has been implicated as contributing to the CSC phenotype is the

myofibroblast. These cells are prominent in the stroma of colorectal tumours and secrete

hepatocyte growth factor (HGF), which appears to be the mediating factor in

dedifferentiation of colorectal cancer cells to CSCs [79].

6. Feedback loop between CSCs and inflammation

CSCs are recognised as the cause of subsequent cancer recurrence following chemotherapy.

These CSCs must possess some mechanism which allows them to re-establish the full

heterogeneity of the tumour in its entirety, a phenomenon that has been observed in xenograft

models. In order for these cells to survive they must be able to promote the formation of a

vascular network in order to receive nutrients and oxygen and they must be able to stimulate

tumour cell proliferation. Their efficient cytokine network accomplishes this through the

secretion of IL-8, MCP-1 and RANTES, which promotes proliferation of stromal cells,

including endothelial cells [80]. IL-8 stimulates the activation of VEGFR2, thus promoting

angiogenic activity. As well, an elevated level of bFGF, IL-6, HGF, PDGF-BB, G-CSF and

SCGF-β have been seen in lung CSCs [80]. Chronic inflammation is mediated by IL-6 and

IL-8 [81], suggesting that CSCs can mediate the inflammatory process. In addition, RANTES

and MCP-1 attract tumour infiltrating lymphocytes [82]. Cross-talk has also been

documented as occurring between basal-like breast cancer cells and monocytes/macrophages

[83]. These basal-like breast cancer cells induced the differentiation of monocytes to

macrophages and stimulated their migration. In addition, several cytokines were shown to be

upregulated in the co-culture of cancer cells and macrophages. These included MCP-1, MCP-

3 and IL-1β [83]. Higher IL-6 mRNA levels have also been observed in mammospheres

correlating IL-6 levels with Notch3 dependent upregulation of Jagged1 and promotion of

CSC features [77, 84]. Additional studies have shown that following metastasis to the lung,

breast cancer cells secreted TGF-β2/ TGF-β3 which induced stromal fibroblasts to express

periostatin which induced recruitment of Wnt ligands, thus providing a positive feedback

mechanism to upregulate Wnt signalling in CSCs [85]. In prostate cancer, a hypoxic

environment led to the increased expression of VEGF and IL-6, again linking these cytokines

secreted by the prostate CSCs to an inflammatory environment [86]. These studies indicate

that the communication between tumour cells and the tumour environment is two-way, with

the CSCs secreting factors that attract the requisite cells into their locale allowing them to set

up ‘home’ in a ‘warm and friendly’ neighbourhood.

7. Epigenetics, Inflammation and CSCs

Epigenetics refers to the heritable changes that occur in gene expression which do not involve

changes in DNA sequences or copy number. Specifically, epigenetic changes may involve

adding or removing various ‘marks’, such as DNA methylation, on the chromatin, and it

reflects the causal relationship between genes and their products, thus affecting their

phenotype. Indeed, damage to cells by increasing levels of reactive oxygen species targets a

complex of transcriptional repressor proteins which predisposes to abnormal DNA

methylation, thus linking inflammation to epigenetics [87]. EZH2, the enhancer of zeste

homolog 2, is a histone methyltransferase that regulates embryonic development and has

been linked to cancer [88]. Its overexpression has been shown to be induced by IL-6 and

TGF-β, with IL-6 activating chromatin-modifying enzymes which can thus resemble p53-

deficeint cells [89, 90]. Indeed, IL-6 driven epigenetic changes have been associated with

CSC features in breast cancer cells [89], and EZH2 levels have been shown to be higher in

breast and pancreatic CSCs as compared to non-CSCs [91]. Src is a non-receptor tyrosine

kinase that has been correlated with advanced malignancy and poor prognosis in a variety of

cancers [92]. Following a transient activation of this protein, an epigenetic switch initiates

and maintains human mammary epithelial cells in a transformed state, a response that is

mediated by the inflammatory NF-κB pathway. This begins a positive feedback loop that

activates Lin28 transcription, reduces Let7 expression which results in higher levels of IL-6,

and through IL-6, activates Stat3 and further stimulates NF-κB [93]. A further study on

microRNA has shed light on this epigenetic switch and other pathways involved in the initial

activation of NF-κB. The microRNAs miR-21 and miR-181b-1 are directly activated by

Stat3. However, it has been shown that either of these microRNAs are able to induce cellular

transformation, and even with the transient expression of these microRNAs lasting only a few

days, the MCF-10A cells were able to be passaged for at least 30 days without losing their

ability to form colonies in soft agar [94]. The induction of either of these microRNAs led to

the direct suppression of the phosphate and tensin homolog (PTEN) and cylindromatosis

(CYLD) tumour suppressor genes, which activated NF-κB. These results have shown that

Stat3, as well as being a downstream effector of IL-6, plays a role in the epigenetic switch

linking inflammation to cancer, alongside miR-21, miR-181b-1, PTEN and CYLD [94].

Linking the tumour microenvironment with cancer development is the loss of tumour

suppressor genes in stromal fibroblasts. The loss of PTEN in stromal fibroblasts promoted

breast cancer progression, including immune cell infiltration and increased angiogenesis [95].

This, and other studies, have suggested that epithelial and stromal cell signalling influences

one another [95-97]. A recent study starts to delineate this process, with a stromal deletion of

Tgfbr2 inducing inflammation, loss of p15 and p16, promoter methylation of p21 and

increased epithelial proliferation leading to squamous cell carcinoma, showing that

inflammation is a critical player in the epigenetic silencing of p21 during tumour progression

[98]. As these epigenetic changes occur in and vary from one cell to another, it is possible

that the specific targeting of these events may represent a new avenue for cancer treatment.

8. Conclusions

Researchers are slowly delineating the origins of cancer. Studies with lineage tracing have

shown, at least in certain tumours, that the origin cell of cancer is linked to stem cells within

the organ and further experimentation suggests that a ‘misplaced’ stem cell could land in the

wrong place following damage to the basement membrane [99, 100]. Once these cells have

invaded the stroma, the immune system detects these ‘foreign’ cells and attempts to eradicate

them. The majority of these cells are destroyed but the accumulation of inflammatory damage

along with these invaders striving to survive, proliferating and developing mutations in the

fight to outwit the immune system, leads to a population expansion of cells that have possibly

lost tumour suppressor genes, such as p53 [100]. Multiple factors in the tumour

microenvironment play a role in tumour cell behaviour. The influx of immune cells adds to

the heterogeneity of tumours with these cells and tumour cells both contributing to the

signalling cascades by secreting cytokines. This inflammation is evident at the earliest stages

of cancer progression and is capable of fostering the growth of small, incipient tumours into

metastatic cancers. Stimuli from tumour cells and immune cells cause cancer cells to

dedifferentiate into cells with the ability to self-renew and to eventually migrate to distant

sites. This is the progression of cancer cells to CSCs and eventually a proportion of these will

progress through the EMT and metastasize. This complex process is slowly being fully

elucidated and this knowledge will assist with the generation of more effective therapies

which will control and may even eradicate cancer in the future.

9. Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The work in the authors laboratories are supported by grants from National Health and

Medical Research Council of Australia, Australia-India Strategic Research Fund and The

CASS Foundation.

References

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Figure legends

Fig. 1. Cancer formation. (A) An initial oncogenic hit provides a cell with the propensity to

form a tumour. (B) Following several more oncogenic hits, a cancer begins following

proliferation of the initial cell. (C) External stimuli, such as cytokines, provides the necessary

environment for cancer cells to dedifferentiate into a cancer stem cell.( D) The primary

tumour continues to grow.( E) Cancer stem cells undergo an EMT and gain the ability to

move from the primary site of tumour formation. (F) These circulating tumour cells home

into a distant site and establish a secondary tumour.(G) The tumour is recapitulated in its

entirety at a secondary metastatic site.

Fig. 2. Inter-related signalling pathways associated with cancer stem cells and the epithelial-

mesenchymal transition.

Fig. 3. Interaction of cancer cells and cancer stem cells with the microenvironment and

immune cells, such as lymphocytes and granulocytes. Red arrows indicate soluble factors

secreted from the tumour that influence the microenvironment; black arrows indicate soluble

factors secreted from the microenvironment that help to sustain and propagate the tumour.