1SCell healing cancer2012.pdf

Throughout adult life there is an ongoing need to produce new cells to replace those that have been lost through normal differentiation or programmed cell death. Many tissues are maintained by stem cells: cells with the capacity for extensive self-renewal and the ability to generate cells that undergo further differentia-tion. In addition to maintaining normal tissue homeo-stasis, the stem cell compartment responds to injury by increasing proliferation and by reducing differentiation until the cellular content of the tissue has been restored1.

Although the body has evolved very effective tissue-repair mechanisms, a close association between chronic tissue damage, inflammation and cancer has been observed2. Tumours can develop, albeit infrequently, at the site of chronic skin wounds or untreated mouth ulcers3. There is a well-established link between ulcerative colitis and colorectal cancer4. Chronic liver inflamma-tion owing to viral hepatitis or excess alcohol consump-tion predisposes to hepatocellular cancer5. Patients with Wilson’s disease or haemochromatosis, which are genetic diseases in which abnormal accumulation of copper or iron causes chronic liver injury, are also at risk of develop-ing liver cancer6. In the stomach, chronic gastritis caused by Helicobacter pylori infection is linked to cancer devel-opment7, and there are several case reports of lung metas-tases at sites of accidental trauma8. Such associations, together with similarities in the histology of wounds and tumours, led Dvorak to the often-cited conclusion that “tumours are wounds that do not heal” (REF. 9).

In this Review, we discuss the links between stem cells, wound healing and cancer. Both wound repair and can-cer are associated with changes in the microenvironment

to which stem cells are exposed, as there is an influx of immune cells, new blood vessel formation, fibroblast proliferation and extracellular matrix (ECM) remodel-ling. Just as heterogeneity within a given cell type is a characteristic of normal tissues with a stem cell com-partment, cellular heterogeneity within tumours has long been recognized. This has led to the concept that tumour maintenance depends on cancer stem cells (also known as tumour-initiating cells) and raises the ques-tion of whether tumours are derived from normal stem cells1,10. We primarily focus on the skin, because the epi-dermal stem cell populations and steps in wound healing are well characterized. In addition, a range of experimen-tally tractable mouse models of skin cancer is available. Nevertheless, the principles outlined in this Review are likely to apply to any tissue in which chronic damage predisposes to cancer.

The microenvironment of wounds and tumoursThe different phases of wound healing in the skin have been extensively documented. All involve dynamic interactions between epidermal cells, dermal cells and bone marrow-derived cells, leading to rapid wound clo-sure and subsequent tissue repair11,12. Wound-healing responses start directly after injury with the formation of a blood clot that initiates a cascade of events, including inflammation: immune cells arrive at the wound site to prevent infection and to remove debris. This is followed by fibroblast proliferation, ECM remodelling, angiogen-esis and the deposition of new connective tissue, which is known as granulation tissue. Epidermal cells are stim-ulated to proliferate and migrate over the granulation

1Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 ORE, UK.2Present address: Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, New York NY 10461, USA.*These authors contributed equally to this work.Correspondence to F.M.W. e-mail: [email protected]:10.1038/nrc3217

Epithelial stem cells, wound healing and cancerEsther N. Arwert1,2*, Esther Hoste1* and Fiona M. Watt1

Abstract | It is well established that tissue repair depends on stem cells and that chronic wounds predispose to tumour formation. However, the association between stem cells, wound healing and cancer is poorly understood. Lineage tracing has now shown how stem cells are mobilized to repair skin wounds and how they contribute to skin tumour development. The signalling pathways, including WNT and Hedgehog, that control stem cell behaviour during wound healing are also implicated in tumour formation. Furthermore, tumorigenesis and wound repair both depend on communication between epithelial cells, mesenchymal cells and bone marrow-derived cells. These studies suggest ways to harness stem cells for wound repair while minimizing cancer risk.

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170 | MARCH 2012 | VOLUME 12 www.nature.com/reviews/cancer

© 2012 Macmillan Publishers Limited. All rights reserved

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KeratinocytesEpithelial cells in a multilayered epithelium, such as the epidermis.

Squamous cell carcinomaMalignant tumour with elements of interfollicular epidermal differentiation.

PsoriasisBenign skin disorder that affects 2% of the world’s population; characterized by epidermal hyperproliferation and skin inflammation.

Sebaceous glandGland that is associated with the junction between the hair follicle and the interfollicular epidermis; releases sebum that lubricates the skin surface.

Interfollicular epidermis(IFE). Multilayered epithelium of the epidermis that lies between the hair follicles; forms the barrier that protects the skin from the external environment.

tissue to repair the epidermis. Once the epidermis has reformed, keratinocytes and fibroblasts synthesize ECM proteins that are incorporated into the new basement membrane. The process of wound healing in the skin has parallels to the repair of other tissues, notably the transient increase in cell proliferation, the inflamma-tory infiltrate and the repair of the basement membrane. Wounds can also be hypoxic before revascularization.

A clear illustration of the link between skin wound-ing and the development of squamous cell carcinoma (SCC) comes from patients with epidermolysis bul-losa. In epidermolysis bullosa, mutations in genes that encode ECM components or their receptors can lead to the detachment of the epidermis from the underlying dermis, resulting in skin blistering and a wound-repair response. Patients with recessive dystrophic epidermo-lysis bullosa (RDEB) have mutations in COL7A1, which encodes the epidermal basement membrane protein type VII collagen, and these patients are at an increased risk of developing SCC. The mechanisms underlying the pathophysiology of RDEB are beginning to be elu-cidated13,14. The similarities between the microenviron-ment of a healing skin wound and an epidermal SCC are summarized in FIG. 1.

A key difference between wound healing and cancer is that wound healing is a self-limiting process; whereas, tumours continue to expand, evolve and spread. This difference is correlated with differences in components of the microenvironment. In wound healing, inflam-mation resolves once re-epithelialization is complete, but this is not the case during tumorigenesis. The con-tribution of inflammation to cancer, which was first demonstrated on injection of Rous sarcoma virus into chicks15,16, has been extensively documented. In chick-ens infected with the virus, tumours form at the site of virus injection and at sites of subsequent wounding. Tumour development at wound sites can be prevented by blocking inflammation. There are striking similarities between the growth factors, cytokines and chemokines that are present in healing wounds and those present in tumours17, but the kinetics of expression differ (TABLE 1). In solid tumours, the same signalling pathways that are transiently upregulated to repair wounds are hijacked and activated constitutively.

Changes in epithelial cells and fibroblasts that are transient during wound healing can be sustained in tumours. The increase in epithelial cell migration and proliferation that is required for wound healing returns to normal on wound closure as the basement membrane is rebuilt. However, in tumours, those processes can con-tinue unchecked; epithelial cells sustain oncogenic muta-tions that can result in immortalization, and they may undergo an epithelial–mesenchymal transition (EMT), which is associated with gaining the properties of cancer stem cells18. One unfavourable outcome of wound heal-ing is that fibroblasts deposit excess collagen, in a process that is known as fibrosis, which leads to scar formation. Fibrotic connective tissue constitutes a highly permis-sive environment for tumour formation19. The wound-healing response of fibroblasts has many similarities to the activation of fibroblasts in the tumour stroma — for example, the expression of markers of myofibroblasts — and can be used to predict the outcome of cancers of the lung, stomach and mammary gland20.

Just as the events during normal wound healing are not sufficient to trigger tumour formation, neither is the chronic inflammation and epidermal hyperproliferation that are features of psoriasis. When six key hallmarks of cancer10,21 are compared with hallmarks of wound heal-ing, it is clear that unique distinguishing features of a tumour include the accumulation of oncogenic changes and the ability to invade the surrounding tissue (TABLE 2).

Epidermal stem cells and wound healingOne of the most important conclusions from recent research on the epidermis is that there are multiple populations of stem cells residing in different locations within the tissue (FIG. 2). There is evidence that under normal homeostatic conditions the stem cells in differ-ent locations maintain the differentiated lineages that are appropriate for those locations. Thus, hair follicle stem cells maintain the hair lineages, sebaceous gland stem cells produce differentiated sebocytes and stem cells in the interfollicular epidermis (IFE) give rise to the outermost barrier layers of the epidermis. However, in response to injury or genetic manipulation, different stem cell pop-ulations are functionally interconvertible. For example, stem cells in the interfollicular epidermis can be repro-grammed to become hair follicle stem cells on sustained activation of the WNT pathway22–24. Although trans-plantation of disaggregated epidermal stem cells into host recipient mice has been used extensively as a stem cell assay (BOX 1), lineage tracing to follow stem cell fate under homeostatic conditions reveals that what stem cells are capable of doing and what they do normally can be quite different. It has been shown, for example, that stem cells that give rise to the IFE and sebaceous gland lineages in undamaged skin form hair follicles on transplantation25.

One of the locations of epidermal stem cells is a region of the hair follicle that is termed the bulge — this is defined as the attachment site of the arrector pili muscle (FIG. 2). The first marker that was used to identify stem cells in the bulge was long-term retention of a DNA label, such as tritiated thymidine or 5-bromo deoxyuridine (BrdU).

At a glance

•Woundhealingandtumorigenesisaretwoprocessesthatrelyonsimilarmolecularmechanisms.Repairoftissueinjuryisaself-limitingprocess;whereas,tumourformationischaracterizedbythecontinuousactivationofthepathwaysinvolved.

•Theinterplayofdifferentcelltypes,suchasepithelial,mesenchymalandimmunecells,isofmajorimportanceinbothwoundrepairandtumourformation.Changesinthemicroenvironmentcausedbytissueinjurycanpermitthedevelopmentofa tumour.

•Stemcellscontributetowoundhealingandtumourformation.Ineachcase,stemcellscanadoptanewlocationthatdiffersfromtheirlocationinundamaged tissue.

•Severalcrucialpathways,suchasHedgehogandWNTsignalling,arederegulatedinwoundhealingandtumorigenesis.DeregulatedHedgehogsignallingislinkedtothedevelopmentofbasalcellcarcinoma;whereas,aberrantWNTsignallingcanresultinavarietyofepidermal tumours.

•Non-dividing,differentiatinganddyingepithelialcellscaneitherpositivelyornegativelyinfluencetumourformation.

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NATURE REVIEWS | CANCER VOLUME 12 | MARCH 2012 | 171


Nature Reviews | Cancer

Cytokinese.g. IL-1α, IL-6 and TNF

Cytokines andgrowth factorse.g. TGFβ, EGF, IL-1 and TNF

Cytokines, MMPs and growth factorse.g. TGFβ, EGF, FGF2,PDGF and IL-1α

Cytokines, MMPs and growth factorse.g. TGFβ, EGF, FGF2,PDGF and IL-1α

Cytokines and growth factorse.g. TGFβ, EGF, FGF2, PDGF and IL-4

Growth factorse.g. FGF2 and VEGF

Growth factorse.g. FGF2 and VEGF

Wound healing (invading edge) Tumour and invading cellsa b

Cytokines and growth factorse.g. TGFβ, EGF, FGF2, PDGF and IL-4

Cytokines andgrowth factorse.g. TGFβ, EGF, FGF2, PDGF and IL-4

Fibrin clot

Bloodvessel

Basementmembrane

Bone marrow-derived inflammatory cells

Extracellular matrixe.g. collagen type III

Apoptotic or necrotic cell

Stem cell

Differentiated cell

Squame

Dividing cell

Endothelial cell

Pericyte

FibroblastsFibrin clot with platelet plug

Other bone marrow- derived cells

PGE2

HMGB1 HMGB1

Growth factorse.g. TGFβ, FGF2 and VEGF

Growth factorse.g. TGFβ, FGF2 and VEGF

Growth factorsand MMPse.g. TGFβ, PDGF,VEGF, MMP1 and MMP2

Growth factorsand MMPse.g. TGFβ, PDGF,MMP1, MMP2and VEGF

PGE2

On wounding, these label-retaining cells move upwards out of the follicle and participate in re-epithelialization26. The first molecular marker of the bulge to be identified was keratin 15 (K15; also known as KRT15). Lineage tracing of hair follicle-derived cells during wound healing, using a truncated K15 promoter to express β-galactosidase as a reporter, showed that the progeny of K15+ bulge cells migrate from the hair follicles into the wound edges and towards the wound centre27. These

studies also showed that, whereas the progeny of K15+ bulge cells make a long-term contribution to the hair follicle lineages, their contribution to the IFE is only temporary, as they are lost from the IFE several weeks after wound healing is completed27.

In recent years, several additional markers of the bulge have been described, including CD34, SOX9 and leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) (FIG. 2). Although there is substantial

Figure 1 | Comparison of the microenvironments of a healing wound and an invading tumour margin. Wound healing (part a) and tumorigenesis (part b) are dynamic events that require interactions with a wide variety of different cell types, including epithelial cells, fibroblasts, endothelial cells and immune cells. Chemokines and cytokines that are released from epithelial cells during injury are very similar to the ones found in invading tumours. In a wound, damage to the skin activates platelets and the formation of a clot. Platelets release a wide range of growth factors and chemo-attractants to recruit immune cells and to develop the granulation tissue by the activation of fibroblasts via transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). Activated fibroblasts can stimulate angiogenesis, as well as the recruitment and activation of inflammatory immune cells. Necrotic and apoptotic cells release high-mobility group protein B1 (HMGB1) and prostaglandin E

2 (PGE

2) that can stimulate epidermal cell proliferation. Similar signalling pathways are

activated in invading tumours. Growth factors and cytokines are produced by epithelial tumour cells in a paracrine and an autocrine manner, resulting in growth, invasion and recruitment of an inflammatory infiltrate in the tumour. Necrotic cells in the tumour core and tumour cells that are dying owing to chemotherapy provide HMGB1 and PGE

2. EGF, epidermal growth

factor; FGF2, fibroblast growth factor-2; IL, interleukin; MMP, matrix metalloproteinase; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.

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IsthmusThe region of the hair follicle that extends between the bulge and the sebaceous gland.

Junctional zoneJunction between the hair follicle, sebaceous gland and infundibulum; the location of LRIG1+ stem cells.

overlap in the expression of the different bulge mark-ers, lineage tracing suggests that they differ in their contribution to wound healing. LGR5+ cells lie in the lower bulge and, in contrast to cells of the upper bulge, are not obligatorily slow cycling (FIG. 2). On wounding, LGR5+ cells migrate upwards into the IFE and sebaceous glands. Unlike the progeny of K15+ bulge cells, the prog-eny of LGR5+ cells can remain in the IFE for more than 1 year post-wounding28. The progeny of SOX9+ cells are reported to permanently contribute to the IFE following wounding29,30.

There are several possible explanations for the appar-ent contradiction that LGR5+ cells and SOX9+ cells provide a long-term contribution to the IFE, whereas K15+ cells do not. There could be technical issues, as the LGR5 and SOX9 studies involved expressing Cre under the control of the endogenous promoters; whereas, the K15 promoter was part of the upstream sequence of

the gene. It is also apparent that different mouse lines can show differences in the efficiency of Cre activa-tion. A further possibility is that the LGR5+ and SOX9+ progeny that provide a long-term contribution to the IFE are a small subpopulation of bulge cells and could even be K15–. Clearly further work is required to resolve these issues.

Whereas in the past the bulge was believed to be the primary residence of hair follicle stem cells, in recent years it has become clear that stem cells also reside above the bulge, in the isthmus (these cells are LGR6+ and/or placenta-expressed transcript 1 (PLET1; also known as C11ORF34 and MTS24)+)31,32, as well as in the junctional zone (leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1)+ cells)25. On wounding, the progeny of LGR6+ stem cells contribute to epidermal repair, as they are recruited into the IFE where they can differentiate into multiple epidermal cell lineages and actively contribute

Table 1 | Cytokines, chemokines and growth factors that influence wound healing and tumour progression*

Cytokines, chemokines and growth factors

Receptors Functions in wounds Functions in cancer Refs

Growth factors

EGF family (EGF, TGFα, HB-EGF, amphiregulin and heregulin)

EGFR, ERBB2 and ERBB4

Epidermal and mesenchymal regeneration; accelerates wound healing

Cancer cell invasion, macrophage signalling and autocrine growth of tumour cells

113–115

FGF family (FGF2) FGFR1 and FGFR2 Early angiogenesis, fibroblast proliferation and re-epithelialization via keratinocyte migration

Angiogenesis and fibroblast proliferation

116–119

TGFβ family TGFβR1 and TGFβR2

Attracts neutrophils and macrophages, mediates ECM deposition, angiogenesis, epithelial cell migration and wound healing

Tumour development, tumour cell invasion and metastasis

120–125

PDGF PDGFR Attracts neutrophils and macrophages, and mediates ECM deposition and angiogenesis. Stimulates wound healing when applied topically

Recruits inflammatory infiltrate and mediates angiogenesis and lymphangiogenesis

126–129

VEGF VEGFR1–3 Angiogenesis Tumour cell invasion and angiogenesis

118,130, 131

Cytokines and chemokines

IL-1α and IL-1β IL-1R Fibroblast and keratinocyte proliferation and neutrophil recruitment

Tumour cell proliferation, angiogenesis and inflammation

82,132

IL-6 IL-6R Fibroblast proliferation and neutrophil recruitment

Tumour development, tumour cell invasion and metastasis

133,134

TNF TNFR1 and TNFR2 Leukocyte infiltration Tumour promotion or suppression 73,113, 135,136

CSF1 CSF1R Recruitment of macrophages and re-epithelialization

Tumour cell invasion and migration

138–140

CCL2 (also known as MCP1)

CCR2, CCR4, UL12, D6 and duffy

Macrophage recruitment, re-epithelialization, angiogenesis and ECM production

Monocyte recruitment, tumour cell invasion and metastasis

140,141

CXCL1 (also known as GROα and KC)

ECRF3, KSHV, duffy and CXCR2

Neutrophil infiltration, epithelial migration and neovascularization

Angiogenesis, invasion and migration

141,142

CXCL2 (also known as MIP2α and GROβ)

CXCR2 Epithelial proliferation Recruits inflammatory infiltrate and migration

143,144

CXCL8 (also known as IL-8) CXCR1, duffy and KSHV

Inflammation, wound contraction and epithelial proliferation

Angiogenesis, migration and invasion

145,146

CXCL12 (also known as SDF1α)

CXCR4 and KSHV Angiogenesis Migration, invasion and angiogenesis

147–149

CSF1, colony stimulating factor 1; CSF1R, CSF1 receptor; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, EGF receptor; FGF, fibroblast growth factor; FGFR, FGF receptor; HB-EGF, heparin-binding EGF-like growth factor; IL, interleukin; MCP1, monocyte chemoattractant protein 1; MIP2α, macrophage inflammatory protein 2α; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; SDF1α, stromal cell-derived factor 1α; TGF, transforming growth factor; TGFβR, TGFβ receptor; TNF, tumour necrosis factor; TNFR, TNF receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. *The cytokines, chemokines and growth factors were included on the basis that they have been shown to influence both wound healing and tumour invasion or progression in vivo.

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Basal cell carcinoma(BCC). Very common, slow-growing epidermal tumour that lacks differentiated cell markers and that is believed to arise from hair follicles.

InfundibulumThe part of the hair follicle that lies above the sebaceous gland and that is continuous with the interfollicular epidermis.

to de novo hair follicle formation31. A further stem cell population that is present in the bulge and the isthmus is defined by the expression of GLI1, which is a transcrip-tion factor that is activated by sonic hedgehog (SHH)33. In these cells, the Hedgehog pathway is activated by SHH, which is produced by sensory neurons that are in contact with the hair follicle. GLI1-expressing cells migrate to the IFE on wounding, where they can make a long-term contribution to the re-epithelialized epidermis33,34.

The hair follicle is not the only location of epider-mal stem cells. Stem cells in the sebaceous gland have been identified on the basis of B lymphocyte-induced maturation protein 1 (BLIMP1; also known as PRDM1) expression35 (FIG. 2), and lineage-tracing experiments have established the existence of cells in the basal layer of mouse IFE36 that maintain the IFE over many months. Mice with mutant EDAR-associated death domain (Edaradd) lack hair follicles in tail skin, but wound closure still occurs, pointing to the dispensa-bility of hair follicle stem cells for wound healing 37. Nevertheless, epidermal wound repair is accelerated during the growth phase (anagen) of the hair growth cycle, a stage in which the hair follicle stem cells are activated, and Edaradd-mutant mice exhibit delayed wound healing37.

In addition to being used for lineage tracing, stem cell markers can also be used to selectively ablate the cells that express them or to delete the markers themselves. For example, ablation of K15+ bulge cells causes the loss of hair follicles, but the IFE remains viable and cutaneous wounds are fully repaired27. On deletion of the bulge stem cell marker SOX9 in adult skin, the hair growth cycle is severely impaired30, and wound repair is severely compromised when Sox9 is deleted in neonatal skin29. The transcription factor LIM homeobox protein 2 (LHX2), which is required for maintenance of the hair follicle stem cell compartment38, differentially regulates SOX9, LGR5 and transcription factor 4 (TCF4; one of the effectors of the WNT pathway)24,39. Most of the bulge cells that proliferate in response to wounding are LHX2+ and wound re-epithelialization is delayed in Lhx2+/– mice. LHX2 positively regulates the expression of SOX9 and TCF4 in bulge cells to promote wound re-epitheli-zation, and negatively regulates LGR5 expression in the secondary hair germ to inhibit the hair follicle cycle39.

With the caveat that one must be cautious when comparing data from different models of wound repair, involving different sized wounds and different ages of mice, it does appear that populations of stem cells differ in their contribution to epidermal repair. The underly-ing mechanisms remain to be determined, however. The differences in contribution to wound healing may reflect intrinsic differences between different stem cells, such as the transcription factors they express, or different properties of stem cell niches, so that they facilitate the maintenance of some stem cells but not others.

Stem cells and wound-induced tumoursBasal cell carcinoma. The most common type of skin cancer in the United States and northern Europe is basal cell carcinoma (BCC), which arises from the inap-propriate activation of Hedgehog signalling40 (FIG. 3). The Hedgehog pathway has an important role in the normal hair growth cycle41 and deletion of Gli2 or Shh results in defective embryonic hair follicle develop-ment42,43. The overexpression of SHH in mouse skin is sufficient to induce BCC formation44. It has long been hypothesized that BCC arises from cells of the hair follicle, partly because of their location and histology and also partly because, in irradiated mouse skin that lacks one allele of the SHH receptor patched 1 (Ptch1), BCCs preferentially arise during anagen — a stage that is marked by the activation of hair follicle stem cells45–47.

In mice, the Hedgehog pathway can be activated in several different ways, including deletion of Ptch1, over-expression of Gli1 or Gli2, or mutational activation of the signalling effector smoothened (Smo) (FIG. 3). When these genes are targeted using the K5 or K14 promoters, which are expressed in stem cells in the IFE, hair follicle and sebaceous gland, BCC-like lesions form44,48–51.

BCCs can arise from multiple stem cell popula-tions, including the bulge and the IFE47. The originating cell can influence the subtype of BCC that develops47,50 and can also affect the likelihood that a tumour will form. By expressing a constitutively activated mutant of SMO in specific subsets of cells, Youssef et al.50 found that BCCs arose preferentially from cells in the IFE and the infundibulum rather than from cells in the bulge. Although SHH is not upregulated in keratinocytes during wound healing34, wounding is necessary for bulge stem cells with an activating SMO mutation to form a tumour51. In a different study, the progeny of LGR5+ cells were found to contribute to BCC formation in the presence or the absence of wounding, but wounding was necessary for BCC to arise in the IFE49. In p53-null epidermis, X-ray-induced BCC in Ptch1+/– mice arises in the IFE as a result of enhanced SMO expression52.

Several conclusions can be drawn from these stud-ies. One is that BCC can have multiple origins. Second, wounding promotes BCC formation, and this can be associated with the mobilization of stem cells out of their normal bulge microenvironment into the IFE27,49,51. The movement of stem cell progeny to a new location can also be induced by the tumour promoter TPA, which stimu-lates bulge cells to move into the IFE and to form BCC on activation of Hedgehog signalling49,51. Last, lineage

Table 2 | Comparison between the hallmarks of cancer and wound healing

Cancer Wound healing

Sustained proliferative signalling Transient proliferative signalling

Evasion of growth suppressors Transient evasion of growth suppression

Invasion and metastasis Activation of cell migration without invasion or metastasis; basement membrane repair*

Enabling replicative immortality No

Inducing angiogenesis‡ Yes

Resisting cell death Transient decrease in terminal differentiation; transient increase in cell death

*The absence of invasion and metastasis in wound healing is linked to the absence of epithelial–mesenchymal transition. ‡In addition to angiogenesis, lymphangiogenesis is stimulated in both cancer and wound healing.

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α6 and β1 integrins

GLI1

GLI1

Junctional zone

Isthmus

Club hair

Hair germ

Dermal papilla

Bulge

Arrector pilli muscle

Sebaceousgland

Infundibulum

BLIMP1

Interfollicular epidermis

• LRIG1• PLET1

CD34

LGR6

• LGR5• K15• SOX9

• K15• SOX9

PilomatricomasBenign skin tumours with elements of hair follicle matrix differentiation.

TrichofolliculomasBenign skin tumours with multiple elements of hair follicle differentiation.

PapillomasBenign tumours with elements of interfollicular epidermal differentiation that can convert into squamous cell carcinomas.

Full-thickness woundingWounding that extends through all the layers of the skin (epidermis, dermis and subdermal fat layer).

tracing shows that individual BCCs are not obligatorily clonal in origin, which should temper the current enthu-siasm for finding the ‘definitive’ tumour-initiating cell within a tumour49,50,53.

Hair follicle and sebaceous gland tumours. Whereas deregulated Hedgehog signalling is linked to the forma-tion of BCC, deregulated WNT signalling (FIG. 3) occurs in different epidermal tumour types. In humans, activat-ing mutations in β-catenin have been found in pilomatri-comas and trichofolliculomas; whereas, mutations in the amino terminus of lymphoid enhancer-binding factor 1 (LEF1) that block β-catenin binding are found in human sebaceous gland tumours23,54,55. Inducible activation of β-catenin in adult mouse epidermis under the control of the K14 promoter leads to the formation of lesions resembling pilomatricomas that regress when β-catenin is no longer activated56. Expression of ΔNLEF1 under the control of the K14 promoter results in the for-mation of sebaceous gland tumours57,58. Ablation of β-catenin expression via the K14 promoter results in the regression of chemically induced papillomas59.

Just as in the case of HH signalling, different epider-mal stem cell populations exhibit differing sensitivity to WNT-associated tumour formation. Sustained activa-tion of β-catenin, under the control of the K15 promoter, stimulates proliferation and the expression of WNT-target genes in the bulge but it is not sufficient to induce pilomatricomas, even when combined with wound-ing60. By contrast, prolonged activation of β-catenin under the control of a truncated K5 (ΔK5) promoter, which is expressed in the sebaceous gland and the hair follicle bulb, leads to the conversion of the sebaceous gland into hair follicles, which subsequently overgrow and resemble benign tumours60. There is some evidence that the WNT pathway is activated during skin wound healing61–64 (FIG. 3).

Papillomas and squamous cell carcinomas. A clas-sic technique for inducing skin tumours is two-stage chemical carcinogenesis. Typically, mice are treated once with DMBA to induce HRAS mutations and they subsequently receive repeated applications of TPA. Although activating RAS mutations have been reported in human SCC, most of these tumours have increased levels of active RAS in the absence of RAS mutations65. Papillomas and SCCs exhibit elements of IFE differentia-tion66. When oncogenic KRAS is targeted to bulge stem cells (via the K15 or K19 promoter) or to the IFE, pap-illomas form; p53 loss results in malignant conversion to invasive SCCs67,68. However, in serial transplantation experiments, high integrin expression (which is a marker for multiple stem cell populations) enriches for tumour-initiating cells in DMBA- and TPA-treated skin, whereas the bulge marker CD34 does not enrich for these cells69. Together with the studies of deregulated Hedgehog and WNT signalling, these observations support the conclu-sion that epidermal tumour type primarily depends on the underlying genetic lesion rather than on the specific stem cell population that has been targeted67.

Just as in BCC, wounding can stimulate epidermal tumour formation in response to RAS pathway activa-tion. The expression of the RAS activator SOS under the control of the K5 promoter gives rise to wound-induced tumours70. When HRAS is expressed under the control of promoters that are expressed in a minority of IFE basal cells, tumours only form on wounding71,72. By contrast, when HRAS is expressed using the K5 promoter, papillo-mas and SCCs can develop spontaneously73. When con-sidering how wounding affects the ability of stem cells to found tumours, it is interesting to note that differences in the susceptibility of different strains of inbred mice to DMBA- and TPA-induced carcinogenesis correlate with differences in their susceptibility to develop tumours on DMBA initiation followed by full-thickness wounding74.

Wounding and TPA treatment result in epidermal hyperproliferation. Thus, in addition to changing the location of stem cell progeny, these stimuli could poten-tially contribute to tumour development by increasing the size of the stem cell compartment. There is evidence that the genetic modifiers that influence strain-specific differences in tumour susceptibility75 also affect the size of the stem cell compartment76. In skin tumours

Figure 2 | Different stem cell compartments in adult mouse back skin. Within the interfollicular epidermis the stem cells express high levels of integrin extracellular matrix receptors, but it is not clear whether the cells are clustered or distributed as single cells, or whether all basal layer cells are stem cells. There is also some uncertainty about the relative distribution of stem cell markers (denoted by coloured cells in the figure) between the junctional zone and the isthmus. Within the bulge, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ cells have a more restricted distribution (closer to the dermal papilla) than keratin 15 (K15)+, CD34+ and SOX9+ cells. The follicle shown is in the resting phase of the hair growth cycle. BLIMP1, B lymphocyte-induced maturation protein 1; LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; PLET1, placenta-expressed transcript 1.

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KeratoacanthomasLow-grade squamous cell carcinomas in skin.

derived from DMBA- and TPA-treated mice, a ninefold increase in cells expressing the bulge marker CD34 was observed59. In the case of junctional zone stem cells, β-catenin activation increases the size of the stem cell compartment25.

The contribution of other cell populationsDifferentiating or dying epidermal cells. The contribu-tion of K15+ and LGR5+ cells to tumour formation is consistent with the view that only stem cells, as long-term residents in the epidermis, have the ability to accu-mulate the genetic lesions that are necessary to found a tumour66,77. Nevertheless, it is clear that non-dividing, differentiating epidermal cells can either positively or negatively contribute to tumour formation78,79. This is illustrated by studies in which different integrin ECM receptors are expressed under the control of the involu-crin promoter, which is specifically active in the supra-basal differentiating cell layers, in order to mimic the suprabasal integrin expression that is seen in hyper-proliferative human skin. These mice do not develop spontaneous tumours. However, suprabasal expres-sion of specific integrins exerts a positive or negative effect on susceptibility to DMBA- and TPA-induced carcinogenesis80.

One of the major pathways that is deregulated on suprabasal expression of β1 integrins is the ERK MAPK cascade. Whereas, suprabasal β1 integrin expression results in sporadic epidermal hyperproliferation and inflammation, the expression of an activated form of the MAPKK MEK1 via the involucrin promoter (in InvEE-transgenic mice) causes the phenotypes to

become constitutive81. InvEE mice develop papillomas and keratoacanthomas at a high frequency at sites of full-thickness wounds82. To conclusively establish that the proliferative, transgene-negative compartment was actively contributing to tumour formation, chimeric mice were generated, and MEK1 transgene-negative cells were detected by green fluorescent protein (GFP) expression. The frequency of wound-induced tumour formation was similar to that of non-chimeric InvEE mice, and GFP+ cells were fully integrated into the tumours, indicating that tumour formation relied on non-cell autonomous signals82, one of which has been identified as interleukin-1α (IL-1α)81,82.

In the epidermis, apoptotic and necrotic cells can also promote the proliferation of neighbouring cells. Apoptotic cells promote wound healing through the production of prostaglandin E2 downstream of activated executioner caspase 3, caspase 6 and caspase 7 (REF. 78). The growth-promoting effect of apoptotic cells has also been observed during breast cancer regrowth following radiotherapy83. Another means by which dying cells stimulate wound repair and tumour formation is via the secretion of high-mobility group protein B1 (HMGB1), which is the proto-typical damage-associated molecular pattern molecule (DAMP) and which initiates the host immune response. HMGB1 is a chromatin-associated protein that can be passively released by necrotic cells (or actively secreted by immune cells) and it functions as an extracellular signal-ling molecule by binding to several cell surface receptors, including toll-like receptors, to promote wound healing, inflammation and tumour metastasis. HMGB1 release from tumour cells as a result of chemotherapy or radio-therapy, which, like wounding, damage tissue, contributes to changes in the tumour microenvironment84.

Bone marrow-derived cells. The disruption of the epi-dermal barrier is known to trigger inflammation. In the case of wound healing, barrier disruption and inflam-mation are transient. By contrast, sustained perturbation of epidermal gene expression can lead to long-term bar-rier disruption and can result in chronic inflammation, which in turn has been linked to cancer. One example of this is the perturbation of epidermal Notch signal-ling. Activation of Notch signalling stimulates epidermal differentiation85–87, and ablation of NOTCH1 expression in adult skin leads to tumour development88,89. Both activation and deletion of Notch1 disrupt the epider-mal barrier and trigger an inflammatory infiltrate90,91. Epidermal Notch1 deletion stimulates epidermal cells to secrete thymic stromal lymphopoietin (TSLP), which in turn stimulates the infiltration of B cells90,92. Conversely, Notch activation in the basal layer of the epidermis triggers dermal accumulation of T lymphocytes91. The non-cell autonomous effects of inhibiting epidermal Notch signalling stimulate the adjacent epidermal cells, in which the pathway is still functional, to become incorporated into tumours88.

Although there are several examples of how targeting an oncogene or a growth factor to the basal epidermal layer, where the stem cells reside, is a more profoundly tumorigenic stimulus than targeting the differentiated

Box 1 | Assays for stem cells and cancer stem cells

Adulttissuestemcellsarecellsthathavetheabilitytoself-renewthroughoutadultlifeandtogenerateprogenythatundergofurtherdifferentiation.Cancerstemcells,ortumour-initiatingcells,arethoughttoberesponsibleforthemaintenanceandmetastasisofsometumours.Likenormalstemcellstheyareabletoself-renewandtheymayproduceprogenythatexhibitsomedifferentiatedcharacteristics.Threemaintypesofassayareusedtoidentifystemcellsandcancerstemcells.

Lineage tracingTheintroductionofastablegeneticmarkerintoasinglecellallowstheidentificationofalltheprogenyofthatcell.Themarkercanbeintroducedintocellsatrandomoritcanbeexpressedunderthecontrolofaspecificpromoter.Lineagetracingrequireshigh-qualityimageanalysistomonitorallthecellswithinamarkedclone,andcarefulstatisticalanalysisofthedata.

TransplantationPopulationsofcells(typicallyisolatedonthebasisoftheexpressionofspecificmarkers)areassayedforself-renewalanddifferentiationortumourformationfollowingtransplantationintoarecipienthost.Thistechniquemakesitpossibletostudythebehaviourofhumancellsinanin vivosetting.However,becausecellsarenotintheirnormalenvironmenttheymaybehavedifferentlyfromhowtheybehaveinintacttissue.Theuseofimmunocompromisedhostmiceprecludestheanalysisofinteractionsbetweenstemcellsandcellsoftheimmunesystem.

Clonal growth in cultureTheabilityofindividualcellstoself-renewandtodifferentiateincultureismeasured.Thistypeofexperimentalapproachhastheadvantagesofspeed,simplicityandeaseofquantitation.ItisalsopossibletomeasurehowcellsrespondtospecificextrinsicsignalssuchasgrowthfactorsandECMproteins.Disadvantagesincludetherestrictedrangeofdifferentiatedlineagesthatcanbesupportedinculture,andtheinabilitytomaintainhomeostasis(balanceofself-renewalanddifferentiation).

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a

b

SHH

WNT

HH in wound healing HH in skin cancer

WNT in wound healing WNT in skin cancer

GLI

SHH

WNT

Smoothened

Patched

SUFUGLI

DSH

β-catenin

β-catenin

Frizzled

GSK3β APC

Axin or conductin

TCF

β-catenin

• Inhibition of HH in adult skin results in defective hair follicle regeneration• Progeny of GLI1- expressing stem cells provide long-term contribution to regenerated epidermis

• Epidermal activation increases stem cell numbers and promotes hair follicle differentiation in wounded and unwounded skin• Loss of β-catenin in wounds results in accelerated wound healing owing to reduced numbers of fibroblasts • Transgenic mice that overexpress stabilized β-catenin in fibroblasts exhibit hyperplasia on wounding

• Activating mutations in β-catenin occur in human

pilomatricomas and trichofolliculomas• LEF1 mutations that block β-catenin signalling occur in sebaceous gland tumours• Deletion of β-catenin in established DMBA- and TPA- induced tumours results in tumour regression

• Mutational activation of the pathway in basal cell carcinoma• Patched mutations in mice result in higher sensitivity to DMBA- and TPA-induced carcinogenesis

layers, the opposite is found when tumour formation is associated with chronic inflammation. The expres-sion of activated MEK1 in the epidermal basal layer results in epidermal hyperproliferation but not in der-mal inflammation; whereas, inflammation is a promi-nent feature of targeting the differentiated cell layers in the InvEE model82,93. Epidermal overexpression of transforming growth factor-α (TGFα) leads to wound-induced tumours, but the effect is more marked when the suprabasal layers, rather than the basal layer, are tar-geted94,95. Mice in which activated HRAS is expressed in the suprabasal layers are more sensitive to wound-induced tumour formation than mice in which HRAS is expressed in the hair follicles73.

In the InvEE model there is constitutive skin inflam-mation, with elevated numbers of macrophages, neu-trophils, T cells and dendritic cells82. In these mice, wound-induced tumour formation is inhibited by the broad-spectrum anti-inflammatory drug dexametha-sone, by depleting the bone marrow of γδ-T cells or by inhibiting macrophages at the wound site82. The impor-tance of B cells and tumour necrosis factor (TNF) sig-nalling in DMBA- and TPA-induced tumorigenesis has recently been demonstrated96, and B cells are also required

for tumour development when human papilloma virus 16 oncogenes are expressed under the control of the K14 promoter97,98. Although macrophage infiltration is a fea-ture of both wound healing and the production of tumour stroma, among the differences between a pro-tumorigenic and an anti-tumorigenic inflammatory infiltrate are changes in the polarization of macrophages99.

Fibroblasts. Just as bone marrow-derived cells com-municate with the epidermis during wound healing and cancer, the same is true for dermal fibroblasts. In early studies, a paracrine signalling loop was identified in which JUN and JUNB antagonistically control cytokine-regulated fibroblast–epidermal interactions99. In the InvEE model, increased epidermal expression of a T cell activator, CD26 (also known as DPP4), is observed, and wound-induced epidermal IL-1α release stimulates CD26 expression by dermal fibroblasts100. Combined pharmacological blockade of IL-1α and CD26 delays tumour onset and reduces tumour incidence. Although fibroblasts in skin wounds express high levels of CD26, the level of CD26 in the stroma of InvEE and other epi-dermal tumours is low. This could reflect a switch from a pro-inflammatory T helper 1 (TH1) environment in wounds to an anti-inflammatory and pro-tumorigenic TH2 cytokine profile in tumours100.

Transdifferentiation. One further way in which wound healing, stem cells and cancer could potentially be linked is both highly speculative and intriguing. There is lit-tle evidence that lineage conversion from one cell type to another (transdifferentiation) occurs to a substantial extent in adult tissues101. Nevertheless, it can occur at a low frequency; for example, through spontaneous cell fusion. There are several reports that bone marrow-derived cells can convert into fibroblasts, with the fre-quency of this increased in the stroma of tumours or healing wounds102,103. Mesenchymal stem cells have also been reported to undergo transdifferentiation into epi-dermal cells, endothelial cells and pericytes, particularly following wounding104,105.

It has recently been shown that allogeneic bone mar-row transplantation can partially alleviate skin blister-ing in patients with RDEB106. Blister repair requires the expression of type VII collagen and its deposition at the basement membrane zone. In an experimental mouse model of RDEB, there is evidence that bone marrow-derived cells are stimulated to transdifferentiate into epidermal cells through the release of HMGB1 from grafted skin107. Because HMGB1 is released by dying tumour cells84, it is possible that transdifferentiation also occurs in tumours.

Conclusions and perspectivesAlthough we have highlighted similarities between wound healing and tumour development, it is clear that, in experimental mouse models, an increase or a decrease in the rate of wound repair is not a simple predictor of skin tumour susceptibility. For example, inhibition of the transcription factor nuclear factor erythroid 2-related fac-tor 2 (NRF2) — which mediates cellular stress responses

Figure 3 | WNT and Hedgehog pathways. Schematics of pathway components are shown. The roles of the Hedgehog (HH) pathway in skin wound healing33,41 and cancer40,45,112 are compared in the table (part a). The roles of the WNT pathway in skin wound healing60-62,64 and cancer23,54,55,59 are also shown (part b). APC, adenomatous polyposis coli; DSH, dishevelled; GSK3β, glycogen synthase kinase 3β; LEF1, lymphoid enhancer-binding factor 1; SHH, sonic hedgehog; SUFU, suppressor of fused; TCF, transcription factor.

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by regulating the expression of cytoprotective genes — in the epidermis does not impair wound healing but does enhance the formation of DMBA- and TPA-induced skin papillomas108. The disruption of protein kinase Cη (PKCη) expression through genetic deletion leads to impaired wound healing but enhanced tumour formation in response to chemical carcinogens109. Conversely, when the catalytic subunit of telomerase, TERT, is expressed in the basal layer of the epidermis wound healing is increased, as is tumour formation110. Epidermal deletion of TGFβ receptor type 2 (TGFβR2) results in accelerated wound closure and an increased incidence of spontane-ous and DMBA-induced SCCs111. These examples pro-vide grounds for optimism that the link between chronic wounds and cancer development can be broken.

When considering potential ways to break the link, a number of possibilities emerge. One is to discover how the properties of stem cells are altered when they end up in a new location. This information might make it pos-sible to determine whether specific stem cell populations should be targeted or whether the cellular environment

is more important. Whether de-localization of stem cells is a feature of other tissues that develop damage-associated tumours remains to be investigated. A sec-ond avenue that is worth exploring is why some stem cells make a transient contribution to wound healing but others make a permanent contribution to the repaired tissue, as this could be relevant to their relative contribu-tions to tumour formation. Finally, it is of major interest to untangle the mechanisms that suppress the immune response after wound repair, because chronic inflamma-tion, as occurs in non-healing wounds, increases the risk of tumour development.

In conclusion, expansion of stem cell populations and migration of stem cell progeny underlie both wound healing and tumour formation. Efficient wound repair is essential for life, and defective wound repair predisposes to cancer. By identifying the microenvironmental signals that co-ordinate normal wound repair, together with the pathways that control stem cell homeostasis and migra-tion, we should be able to prevent wounds from acting as a stimulus for tumour formation.

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AcknowledgementsThe authors thank I. Brownell, R. Toftgard, K. Kretzschmar and K. Jensen for advice on Figure 2. F.M.W. gratefully acknowledges financial support from the Wellcome Trust, Medical Research Council and Cancer Research UK. E.H. is supported by EUFP7 HEALING network. E.N.A. is a recipient of a Sir Henry Wellcome postdoctoral fellowship.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONFiona M. Watt’s homepage: http://wattlab.cscr.cam.ac.uk

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