Cellular and Molecular Basis ofWound Healing and Diseaseswith a “Wound Signature”

Colin JamoraIFOM-inSTEM Joint Research Laboratory

Institute for Stem Cell Biology and Regenerative MedicineBangalore, India

National Taiwan University Medical SchoolMarch 5, 2014

Anatomy of the Skin

Fuchs and Raghavan Nature Reviews Genetics 2002

Wound

•  Proliferation of skin stem cells •  Angiogenesis •  Wound closure

Proliferation

•  Cell & ECM remodeling

Remodeling

•  Recruitment of immune cells

Inflammation

Phases of the Wound-healing Program

Probing fibroblast biology in the mammalian skin

Activated Fibroblasts in Tissue Repair and Disease

Wound healing

Adapted from: Kalluri and Zeisberg 2006

Koontongkaew 2013 Tissue Fibrosis

Hyperactive

Myofib

roblast

Tumor Stroma

CAF

Inflammation Proliferation Remodeling

Myofibroblast Differentiation in Skin Wounds & Cancer

Scar formation in wounded Snail transgenic skin

Human skin Mouse skin

Snail is involved in development and disease

K14Snail

(Hoot K. E. et al. J. Clin. Invest 2008)

Human cutaneous SCC

High incidence of Snail expression in epithelial cancers

* Snail expression correlates with poor prognosis or relapse

Adapted from KF Becker et al., Cells Tissues Organs 2007 Du et al., 2010

Expression profile of Snail in the skin

Jamora et al., 2004

Mikkola and Millar, 2006

Budding Morphogenesis Mimics Tumor Invasion

Expression profile of Snail in the skin

K14-Snail

Jamora et al., 2004

What is the function of Snail in hair budmorphogenesis and cutaneous disease?Effect of Snail overexpression in the epidermis

Snail expression induces epidermal involution & hyperplasia

WT Snail Tg

Metastasis associated markers are expressed in Snail transgenic epidermis

Snail induces cutaneous inflammation

Characteristics of activated fibroblasts

From Kalluri and Zeisberg 2006

Snail transgenic skin displays elevated dermal ECM

K5/

fibro

nect

inK

5/co

llage

nSnail TgWT

Equilibrium between ECM production and turnover is disrupted

Snail transgenic mouse has thick skin

WT Snail Tg

K14Snail

(Hoot K. E. et al. J. Clin. Invest 2008)

Human cutaneous SCC

Snail expression in cancer & fibrotic disease

Du et al., Cancer Research 2010

in collaboration with John Varga (Northwestern)

Figure 5. Expression of Snail in human sclerotic skin. Immunohistochemistry of normal and sclerotic (Ssc) skin using an antibody specific for human Snail.

scleroderma

How is epidermal Snail inducing dermal fibrosis?

epid

erm

is

Snail

derm

is

Active fibroblasts are not a product of an EMT

Guarino et al., Human Pathology 2009

 

 

Figure 2. Lineage tracing of the activated fibroblasts. Epithelial cells in the skin of wild type (WT) or Snail transgenic mice (K14-Snail) were labeled in blue with X-gal staining when these respective mice were mated with ROSA26/K14-Cre mice. Activated dermal fibroblasts were labeled with an antibody recognizing

αSMA (brown). 

β-ga

l/αSM

A

Primary Dermal fibroblasts

Control CM

Snail CM

WT CM

Snail CM

Assay to test for secreted factor capable of activating dermal fibroblasts

WT keratinocytes

Snail transgenickeratinocytes

untreated WT skin CM

Snail Tg skin CM TGFβ2

% g

el c

ontra

ctio

n

untreated WT skin CM

Snail Tg skin CM TGFβ2

Secreted factor can activate dermal fibroblasts: gel contraction

Conditioned media from Snail epidermal explants can induce expression of active fibroblast genes

0

5

10

15

20

25

mRN

A ex

pres

sion

(rela

tive

fold

)

WT Snail Tg Snail Tg +IL1R KO df

Snail Tg +TGFβ inhib

CTGF

αSMA

Conditioned media

TGFβ and tissue fibrosis

Clinical trials targeting TGFβTargeting the TGFβ signalling pathway in diseaseRosemary J. Akhurst & Akiko HataNature Reviews Drug Discovery 11, 790-811 (October 2012)

11/13/13, 6:04 PMTable 1 : Targeting the TGF[beta] signalling pathway in disease : Nature Reviews Drug Discovery

Page 1 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html

TABLE 1 | Summary of clinical trials for TGFβ inhibitory drugs

FROM THE FOLLOWING ARTICLE:Targeting the TGFβ signalling pathway in disease

Rosemary J. Akhurst & Akiko Hata

Nature Reviews Drug Discovery 11, 790-811 (October 2012)

doi:10.1038/nrd3810

Back to article | Back to figures and tables | Next table

Drug; company Type Targets Diseaseapplications

Stage Clinical trialidentifiers

Summary ofresults

Refs

Trabedersen (AP12009);Antisense Pharma

Antisenseoligo

TGFβ2ligand

Glioblastoma PhaseI/IIb

NCT00431561 Safe 70,73,74

Pancreatic cancer,MetM, coloncancer

Phase I NCT00844064 Pancreatic cancertrials continue

84

Glioblastoma Phase III NCT00761280 Glioblastoma trialsstopped in March2012 owing toadvances instandard of careand neurosurgery(BOX 4)

-

Belagenpumatucel-L(Lucanix); NovaRx

Antisensegene-

modifiedallogeneictumour cellvaccine

TGFβ2 NSCLC Phase III NCT00676507 Well tolerated in 75patients; survival

advantage justifiesfurther Phase IIIevaluation

85,86,87

Disitertide (P144); Digna Peptide Peptide Skin fibrosis in Phase II NCT00574613, Preclinical efficacy 75,76,77

11/13/13, 6:04 PMTable 1 : Targeting the TGF[beta] signalling pathway in disease : Nature Reviews Drug Discovery

Page 2 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html

Disitertide (P144); DignaBiotech

Peptide Peptidebased onTβRIIIthatblocksligandbindingtoreceptors

Skin fibrosis insystemic sclerosis

Phase II NCT00574613,NCT00781053

Preclinical efficacyin peritonealfibrosis associatedwith peritonealdialysis, renal andcardiac fibrosis,corneal haze andretinal AMD; safetyand efficacy inPhase IIa clinicaltrial forscleroderma/skinfibrosis

75,76,77

Lerdelimumab (CAT-152);Cambridge AntibodyTechnology

Humanizedantibody

TGFβ2ligand

Reduction ofscarring afterglaucoma surgery

Phase III(complete)

- Safe; ineffective inreducing scarringin Phase III trial

88,89

Metelimumab (CAT-192);Cambridge AntibodyTechnology

HumanizedAntibody

TGFβ1ligand

Diffuse systemicsclerosis

Phase I/II NCT00043706 Ineffective whensystemicallyadministered indoses up to 10 mgper kg

90

Fresolimumab (GC-1008);Cambridge AntibodyTechnology/Genzyme/Sanofi

Humanizedantibody

TGFβ1,TGFβ2andTGFβ3ligands

Focal segmentalglomerulosclerosis

Phase I NCT00464321 Completed andsafe; plans toprogress

92

Systemic sclerosis Phase I NCT01284322 Still recruiting -Study ongoing -Completed, noresults

-

Myelofibrosis Phase I NCT01291784 See BOX 4 93

IPF Phase I NCT00125385 See BOX 4 93Renal cellcarcinoma

Phase I NCT00356460 See BOX 4 93

Malignantmelanoma

Phase I NCT00356460 See BOX 4 81

Metastatic breastcancer (withradiotherapy)

Phase I NCT01401062 Active andrecruiting patients

-

Relapsed Phase II NCT01112293 Ongoing but not -

What is in secreted by Snail expressing keratinocytes to activate dermal fibroblasts?

epid

erm

isde

rmis

derm

is

Snail? Collagen contraction activity

Induction of CTGF & αSMA

Concanavalin A sepharose columnCollagen contraction activityInduction of CTGF & αSMA

WT or Snail Tg Conditioned media

Anion exchange chromatography (Mono Q)

MassSpec

Candidate proteins mediating epithelial-mesenchymal crosstalk

Spondin-2 is induced in Snail expressing keratinocytes

Figure 6. Snail is sufficient to induce spondin-2/mindin expression. RT-PCR of RNA extracted from wild type (WT) or Snail transfected (Snail) keratinocytes. GAPDH was used as a loading control. Reverse transcriptase (RT) was either added (+) to the reaction or not (-) to control for genomic DNA contamination

spon2

GAPDH

snail

Background on spondin-2 (aka mindin)

What is spondin-2/mindin?•  Member of the F-spondin family•  Secreted, associates with ECM•  Component of basal lamina (zebrafish)•  Ligand for integrins•  PRR – production of inflammatory cytokines•  Promotes outgrowth of hippocampal neurons

Wynn & Ramalingam 2012

Mindin activates NFκB in dermal fibroblasts

dermalfibroblasts

(NFκB)

Control + mindin

0

4

8

12

16

20

Control Mindin

Rel

ativ

e le

vel o

f R

AN

TES

RN

A

macrophages (TAM). Evidence of this is seen in the Snailtransgenic skin by the coexpression of the lectin CD206 on asubset of macrophages (Fig. 2C) and the presence of othermarkers of TAMs (Supplementary Fig. S3B and C). Interest-ingly, it has been reported that TAMs are a source of MMP-9,which is found exclusively in the transgenic dermis (Fig. 1B),and this enzyme contributes to their role in the metastaticcascade (27). The importance of these various immune cells inmanifesting the changes in the Snail transgenic skin wasshown by the ability of an immunosuppressive cocktail tosignificantly reduce the epidermal involution and hyperplasia(Fig. 2D) and cutaneous inflammation (Supplementary Fig. S4)in the mutant mouse.Among these infiltrating immune cells, macrophages have

garnered extensive attention for their remarkable ability topromote tumor proliferation and metastasis (28). Thus, themechanism by which Snail expressing keratinocytes inducestheir recruitment into the skin becomes an important pro-blem to resolve. We observed that the transgenic epidermishad an elevated level of monocyte colony-stimulating factor-1(CSF-1), which is a well-known chemoattractant for macro-phages (Fig. 3A; ref. 29). The induction of CSF-1 seems to becell autonomous, as transfection of Snail into primary kera-tinocytes is sufficient to elicit CSF-1 expression (Fig. 3A).

Importantly, we found, using a Transwell assay, that condi-tioned medium (CM) from epidermal explants of transgenicmice is capable of recruiting macrophages (Fig. 3B). Aninhibitory antibody against this cytokine shows that CSF-1is a required component of the CM to stimulate macrophagemobilization. Because the epidermal explants used to condi-tion the media are a heterogeneous population of cells, wetested whether Snail expression in keratinocytes is directlyinvolved in the recruitment of macrophages by reconstitutingthis process completely in vitro. Primary keratinocytes trans-fected with Snail are capable of synthesizing and secretingCSF-1 to promote macrophage recruitment (Fig. 3C). Thesemacrophages can, in turn, potently stimulate invasion ofprimary keratinocytes through an extracellular matrix(Fig. 3D). Moreover, the cytokine milieu present in the trans-genic skin favors the polarization of the macrophages intoTAMs (Fig. 2B and C; Supplementary Fig. 3B and C), whichhave been localized to areas of metastasis and shown topromote tumor cell invasion (30, 31). Consistent with thisscenario, we found that TAMs can stimulate keratinocyteinvasion at even higher levels than the classically activatedmacrophages (Fig. 3D).

Given TAMs ability to stimulate proliferation of breastcarcinoma cells (32), we hypothesized that they may also

WT

FN

pNFkB

K5

CD3K5

CD206Mac-1

Mac-1K5

epi

epi

epiepi

epi

epi

epi

epi

epi

epi

hf

hf

hf

hf

hfhf

epider

der

der

der

der

der

der

epi

der

epi

der

der

der

der

der

hf

WT

WT

RT

GAPDH

IL-4

IFNγ

IL-13

A B C

D

Snail Tg Snail Tg

Tg + PBS Tg + DI

WT Snail Tg

Figure 2. Inflammation in the Snail transgenic skin. A, expression of the mesenchymal marker fibronectin (FN), activated NFkB (pNFkB), and the panT-cell marker CD3 in WT (left) and Snail transgenic (Snail Tg, right) skin. B, profile of the Th1 cytokine IFNg and Th2 cytokines IL-4 and IL-13 viareverse transcription PCR with GAPDH as a loading control. C, infiltration of macrophages detected by Mac-1 staining in the dermis of WT and Snail Tg skin(top) and the presence of M2/TAMs marked by a subset of macrophages (Mac-1; green) coexpressing the mannose receptor recognized by theCD206 antibody in red. D, histologic analysis of the effect of PBS vehicle control or dexamethasone–indomethacin (DI) immunosuppressive cocktail on thetransgenic phenotype. Bars, 30 mm.

Role of Snail in the Skin

www.aacrjournals.org Cancer Res; 70(24) December 15, 2010 10083

American Association for Cancer Research Copyright © 2010 on October 12, 2011cancerres.aacrjournals.orgDownloaded from

DOI:10.1158/0008-5472.CAN-10-0324

WT Snail Tg

Spondin-2/mindin activates NFκB in dermal fibroblasts

0

5

10

15

20

25

Control Spondin-2

Act

ivity

of N

FκB-

luc

repo

rter

Intrinsic Gene Expression Subsets of DiffuseCutaneous Systemic Sclerosis Are Stable in SerialSkin BiopsiesSarah A. Pendergrass1, Raphael Lemaire2, Ian P. Francis2, J. Matthew Mahoney1, Robert Lafyatis2 andMichael L. Whitfield1

Skin biopsy gene expression was analyzed by DNA microarray from 13 diffuse cutaneous systemic sclerosis(dSSc) patients enrolled in an open-label study of rituximab, 9 dSSc patients not treated with rituximab, and 9healthy controls. These data recapitulate the patient ‘‘intrinsic’’ gene expression subsets described previously,including fibroproliferative, inflammatory, and normal-like groups. Serial skin biopsies showed consistent andnon-progressing gene expression over time, and importantly, the patients in the inflammatory subset do notmove to the fibroproliferative subset, and vice versa. We were unable to detect significant differences in geneexpression before and after rituximab treatment, consistent with an apparent lack of clinical response. Serialbiopsies from each patient stayed within the same gene expression subset, regardless of treatment regimen orthe time point at which they were taken. Collectively, these data emphasize the heterogeneous nature of SScand demonstrate that the intrinsic subsets are an inherent, reproducible, and stable feature of the disease that isindependent of disease duration. Moreover, these data have fundamental importance for the futuredevelopment of personalized therapy for SSc; drugs targeting inflammation are likely to benefit those patientswith an inflammatory signature, whereas drugs targeting fibrosis are likely to benefit those with a fibro-proliferative signature.

Journal of Investigative Dermatology advance online publication, 9 February 2012; doi:10.1038/jid.2011.472

INTRODUCTIONSystemic sclerosis (SSc) is a multisystem autoimmunedisorder with a hallmark of skin fibrosis and thickening alongwith significant internal organ involvement (Mayes et al.,2003). SSc has historically been divided into limited anddiffuse disease based on the extent of skin involvement, withlimited cutaneous SSc (lSSc) involving skin restricted to theregions below the elbows, knees, and face, and diffusecutaneous SSc (dSSc), including more proximal skin. Thedegree of skin involvement has a direct correlation with SScprognosis and internal organ complications (Barnett et al.,1988; Scussel-Lonzetti et al., 2002). However, within dSSc

and lSSc, there is a heterogeneous range of skin and internalorgan involvement. Approaches that objectively quantifydisease heterogeneity and predict internal organ involvementare critically needed.

Previous genome-wide gene expression studies in SSc skinidentified disease-specific gene expression signatures in bothlesional and non-lesional skin biopsies that are distinct fromthose found in healthy controls (Whitfield et al., 2003;Gardner et al., 2006; Milano et al., 2008). In addition, wehave shown that distinct gene expression signatures divideSSc patients into ‘‘intrinsic subsets’’, capturing the clinicalheterogeneity of limited versus diffuse SSc, but extending thisheterogeneity by revealing that patients with dSSc fall intoseveral different subsets based on gene expression in the skin(Milano et al., 2008). These results suggested that distinctpathogenic mechanisms may drive disease in differentpatients or at different stages of the disease. We previouslyidentified four intrinsic gene expression subsets: a ‘‘diffuse-proliferation’’ group comprised completely of patients withdSSc (here referred to as fibroproliferative), showing increasedexpression of genes associated with cell proliferation thatcould be further subdivided into two groups: ‘‘diffuse 1’’ and‘‘diffuse 2’’; an ‘‘inflammatory’’ group comprised of dSSc,lSSc, and morphea samples, showing increased expressionof genes associated with inflammation; a ‘‘limited’’ group

& 2012 The Society for Investigative Dermatology www.jidonline.org 1

ORIGINAL ARTICLE

Received 10 February 2011; revised 22 November 2011; accepted 27November 2011

1Department of Genetics, Dartmouth Medical School, Hanover,New Hampshire, USA and 2Boston University School of Medicine, ArthritisCenter, Boston, Massachusetts, USA

Correspondence: Michael L. Whitfield, Department of Genetics, DartmouthMedical School, 7400 Remsen, Hanover, New Hampshire 03755, USA.E-mail: michael.whitfield@dartmouth.edu; or Robert Lafyatis, BostonUniversity School of Medicine, Medical Campus, Evans 501, 72 East ConcordStreet, Boston, Massachusetts 02118-2526, USA. E-mail: lafyatis@bu.edu

Abbreviations: dSSc, diffuse cutaneous SSc; GO, gene ontology; lSSc, limitedcutaneous SSc; MRSS, modified Rodnan skin score; PPAR-g, peroxisomeproliferation–activated receptor-g; SSc, systemic sclerosis

Functional Group 1Category TermGOTERM_BP_ALL GO:0002376~immune system processGOTERM_BP_ALL GO:0006955~immune responseGOTERM_BP_ALL GO:0050896~response to stimulusFunctional Group 2Category TermGOTERM_BP_ALL GO:0009611~response to woundingGOTERM_BP_ALL GO:0006950~response to stressGOTERM_BP_ALL GO:0009605~response to external stimulusGOTERM_BP_ALL GO:0006952~defense responseGOTERM_BP_ALL GO:0006954~inflammatory responseFunctional Group 3Category TermGOTERM_BP_ALL GO:0032502~developmental processGOTERM_BP_ALL GO:0048513~organ developmentGOTERM_BP_ALL GO:0007275~multicellular organismal developmentGOTERM_BP_ALL GO:0048856~anatomical structure developmentGOTERM_BP_ALL GO:0048731~system developmentGOTERM_BP_ALL GO:0032501~multicellular organismal process

Category TermGOTERM_BP_ALL GO:0006915~apoptosisGOTERM_BP_ALL GO:0012501~programmed cell deathGOTERM_BP_ALL GO:0008219~cell deathGOTERM_BP_ALL GO:0016265~deathGOTERM_BP_ALL GO:0048468~cell developmentGOTERM_BP_ALL GO:0042981~regulation of apoptosisGOTERM_BP_ALL GO:0043067~regulation of programmed cell deathGOTERM_BP_ALL GO:0048869~cellular developmental processGOTERM_BP_ALL GO:0030154~cell differentiation

Category TermGOTERM_BP_ALL GO:0006928~cell motilityGOTERM_BP_ALL GO:0051674~localization of cellGOTERM_BP_ALL GO:0016477~cell migration

Category TermGOTERM_BP_ALL GO:0006817~phosphate transportGOTERM_BP_ALL GO:0015698~inorganic anion transportGOTERM_BP_ALL GO:0006820~anion transport

Category TermGOTERM_BP_ALL GO:0007243~protein kinase cascadeGOTERM_BP_ALL GO:0009966~regulation of signal transductionGOTERM_BP_ALL GO:0009967~positive regulation of signal transductionGOTERM_BP_ALL GO:0007249~I-kappaB kinase/NF-kappaB cascadeGOTERM_BP_ALL GO:0043123~positive regulation of I-kappaB kinase/NF-kappaB cascadeGOTERM_BP_ALL GO:0043122~regulation of I-kappaB kinase/NF-kappaB cascade

Category TermGOTERM_BP_ALL GO:0001944~vasculature developmentGOTERM_BP_ALL GO:0048514~blood vessel morphogenesis

From JID 2012Supp. Fig. 3

Mindin contributes to inflammation

K5 Mac1

K5 Mac1

K5 Mac1

K5 Mac1

K14Snail – Mindin +/- K14Snail – Mindin -/-

K14Snail + Mindin +/- K14Snail + Mindin -/-

05

1015202530354045

WT Snail Snail + mindin KO

# m

acro

phag

es/fr

ame

Snail+ keratinocytes

mindin

dermal fibroblasts

Secretion ofProinflammatory

cytokines

X

Immunosuppression blocks epidermal hyperplasia and involution

WT Snail Tg Snail Tg +anti-inflammatory

K14Snail- Mindin +/- K14Snail- Mindin -/-

K14Snail+ Mindin +/- K14Snail+ Mindin -/-

Mindin contributes to epidermal hyperplasia

010203040506070

WT Snail Snail + mindin KO

epid

erm

al th

ickn

ess (

mm

)

Does mindin contributes to dermal thickening?

Wynn & Ramalingam 2012

Mindin contributes to dermal thickening

0

10

20

30

40

50

60

70

WT Snail Snail + mindin KO

derm

al th

ickn

ess (

mm

)

Snail promotes dermal fibroblast crosstalk

mindin

adapted from Kalluri and Zeisberg 2006

Acknowledgements

Postdocs Brijesh Ajjappala Subhasri Ghosh

Manando Nakasaki (UCSD) Tuan-Lin Tan (Singapore)

Yoshikazu Nakamura (Tokyo) Fei Du (St. Jude’s Hospital)

Students Tanay Bhatt (NCBS, PhD) Sunny Kataria (NCBS, PhD) Neha Pincha (NCBS, PhD)

Federica Centonze (IFOM, MSc)

Jr. Research Fellows Surya Prakash Batta

Syed Abrar Rizvi Edries Hajam

Isha Rana (Research Tech)

Clinical trials targeting TGFβTargeting the TGFβ signalling pathway in diseaseRosemary J. Akhurst & Akiko HataNature Reviews Drug Discovery 11, 790-811 (October 2012)

11/13/13, 6:04 PMTable 1 : Targeting the TGF[beta] signalling pathway in disease : Nature Reviews Drug Discovery

Page 1 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html

TABLE 1 | Summary of clinical trials for TGFβ inhibitory drugs

FROM THE FOLLOWING ARTICLE:Targeting the TGFβ signalling pathway in disease

Rosemary J. Akhurst & Akiko Hata

Nature Reviews Drug Discovery 11, 790-811 (October 2012)

doi:10.1038/nrd3810

Back to article | Back to figures and tables | Next table

Drug; company Type Targets Diseaseapplications

Stage Clinical trialidentifiers

Summary ofresults

Refs

Trabedersen (AP12009);Antisense Pharma

Antisenseoligo

TGFβ2ligand

Glioblastoma PhaseI/IIb

NCT00431561 Safe 70,73,74

Pancreatic cancer,MetM, coloncancer

Phase I NCT00844064 Pancreatic cancertrials continue

84

Glioblastoma Phase III NCT00761280 Glioblastoma trialsstopped in March2012 owing toadvances instandard of careand neurosurgery(BOX 4)

-

Belagenpumatucel-L(Lucanix); NovaRx

Antisensegene-

modifiedallogeneictumour cellvaccine

TGFβ2 NSCLC Phase III NCT00676507 Well tolerated in 75patients; survival

advantage justifiesfurther Phase IIIevaluation

85,86,87

Disitertide (P144); Digna Peptide Peptide Skin fibrosis in Phase II NCT00574613, Preclinical efficacy 75,76,77

11/13/13, 6:04 PMTable 1 : Targeting the TGF[beta] signalling pathway in disease : Nature Reviews Drug Discovery

Page 2 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html

Disitertide (P144); DignaBiotech

Peptide Peptidebased onTβRIIIthatblocksligandbindingtoreceptors

Skin fibrosis insystemic sclerosis

Phase II NCT00574613,NCT00781053

Preclinical efficacyin peritonealfibrosis associatedwith peritonealdialysis, renal andcardiac fibrosis,corneal haze andretinal AMD; safetyand efficacy inPhase IIa clinicaltrial forscleroderma/skinfibrosis

75,76,77

Lerdelimumab (CAT-152);Cambridge AntibodyTechnology

Humanizedantibody

TGFβ2ligand

Reduction ofscarring afterglaucoma surgery

Phase III(complete)

- Safe; ineffective inreducing scarringin Phase III trial

88,89

Metelimumab (CAT-192);Cambridge AntibodyTechnology

HumanizedAntibody

TGFβ1ligand

Diffuse systemicsclerosis

Phase I/II NCT00043706 Ineffective whensystemicallyadministered indoses up to 10 mgper kg

90

Fresolimumab (GC-1008);Cambridge AntibodyTechnology/Genzyme/Sanofi

Humanizedantibody

TGFβ1,TGFβ2andTGFβ3ligands

Focal segmentalglomerulosclerosis

Phase I NCT00464321 Completed andsafe; plans toprogress

92

Systemic sclerosis Phase I NCT01284322 Still recruiting -Study ongoing -Completed, noresults

-

Myelofibrosis Phase I NCT01291784 See BOX 4 93

IPF Phase I NCT00125385 See BOX 4 93Renal cellcarcinoma

Phase I NCT00356460 See BOX 4 93

Malignantmelanoma

Phase I NCT00356460 See BOX 4 81

Metastatic breastcancer (withradiotherapy)

Phase I NCT01401062 Active andrecruiting patients

-

Relapsed Phase II NCT01112293 Ongoing but not -

TGFβ and tissue fibrosis

Failure in clinical trials

untreated WT skin CM

Snail Tg skin CM TGFβ2

% g

el c

ontra

ctio

n

untreated WT skin CM

Snail Tg skin CM TGFβ2

Secreted factor can activate dermal fibroblasts: gel contraction

Snail is sufficient to activate dermal fibroblasts

Tubulin

Snail

untra

nsfe

cted

K14

vec

tor

K14

-Sna

il

Transfection ofPrimary keratinocytes

Effect of CM onDermal fibroblasts (contraction)

0

10

20

30

40

50

Untransfected K14 vector K14-Snail

% g

el c

ontra

ctio

n

CM from transfected keratinocytes

Untreated CM

95oC CM

Cancer associated fibroblasts

483 Am J Cancer Res 2011;1(4):482-497

progression of breast carcinoma [16,17,13]. This large heterogeneity in marker expression for CAFs originating from different tumors may be explained by their possible diverse origin. Indeed, CAFs are variously reported to stem from resident local fibroblasts, bone marrow-derived progenitor cells or trans-differentiating epithelial/endothelial cells through epigenetic transitions (see below) [18,19,20,21]. The role of CAFs in tumor progression is multi-faceted. Similarly to immune cells, which initially repress malignant growth, CAFs inhibit early stages of tumor progression, mainly through the formation of gap junctions between acti-vated fibroblasts [19, 20]. Conversely, later on CAFs become activated by several tumor-secreted factors and promote both tumor growth and progression. Two closely interactive pathways are established in the crosstalk be-tween cancer and stromal cells: a) in the “efferent” pathway, cancer cells trigger a reac-tive response in the stroma, and b) in the “afferent” pathway, the modified stromal cells in

the surrounding microenvironment affect can-cer cell responses [22,23] (Figure 1). The trans-differentiation of CAFs, a process commonly called mesenchymal-mesenchymal transition (MMT) [6], is currently poorly understood. TGF-ơ1 has been largely acknowledged to be one of the major tumor-cell derived factors affecting CAF activation [24]. Nevertheless other pro-fibrotic factors can be released by cancer cells and act on CAFs inducing their activation, in-cluding PDGF-Ơ/ơ [25,26], basic fibroblast growth factor (b-FGF) [27] or interleukin (IL)-6 [23]. Several data indicate that activation of CAFs is under a clear redox control. Tumor growth factor (TGF)-ơ1 causes an increase in reactive oxygen species (ROS) in CAFs, which is responsible for downregulation of gap junctions between CAFs, for their achievement of MF-phenotype, as well as for their tumor promoting activity in skin tumors [28,29]. Antioxidant treat-ments, or the micronutrient selenite, prevent CAF activation and their enhancement of tumor invasion [28]. In keeping, the activation of pros-tate CAFs by tumor-secreted IL-6 is again redox-

Figure 1. Interplay between CAFs and tumor cells. Tumor progression needs a positive and reciprocal feedback be-tween CAFs and cancer cells. Cancer cells induce and maintain the fibroblasts activated phenotype which, in turn, produce a series of growth factors and cytokines that sustain tumor progression by promoting ECM remodelling, cell proliferation, angiogenesis and EMT.

Adapted from Cirri and Chiarugi, Am J. Cancer Res 2011

SNAIL

Mindin

cell proliferationinflammation

Snail expressing keratinocytes activate CAFs

Snail transgenic skin displays markers of cancer stem cells

10/21/12 4:40 PMSlug and Sox9 cooperatively determine the mammary stem ... [Cell. 2012] - PubMed - NCBI

Page 1 of 1http://www.ncbi.nlm.nih.gov/pubmed/22385965

Cell. 2012 Mar 2;148(5):1015-28.

Slug and Sox9 cooperatively determine the mammary stem cellstate.Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, Itzkovitz S, Noske A, Zürrer-Härdi U, Bell G,Tam WL, Mani SA, van Oudenaarden A, Weinberg RA.Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

AbstractRegulatory networks orchestrated by key transcription factors (TFs) have been proposed to play acentral role in the determination of stem cell states. However, the master transcriptional regulators ofadult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that actcooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression ofexogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-termmammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinctautoregulatory gene expression programs. We also show that coexpression of Slug and Sox9promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and isassociated with poor patient survival, providing direct evidence that human breast cancer stem cellsare controlled by key regulators similar to those operating in normal murine MaSCs.

Copyright © 2012 Elsevier Inc. All rights reserved.

PMID: 22385965 [PubMed - indexed for MEDLINE] PMCID: PMC3305806 [Available on 2013/3/2]

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Du et al., 2010