Ldb 145 Geni Mutanti_2014-11-18 Jamora - ricerca scientifica 3

1. Cellular and Molecular Basis of
Wound Healing and Diseases
with a “Wound Signature”
Colin Jamora
IFOM-inSTEM Joint Research Laboratory
Institute for Stem Cell Biology and Regenerative Medicine
Bangalore, India
National Taiwan University Medical Scho
March 5, 2014

2. Anatomy of the Skin
Fuchs and Raghavan Nature Reviews Genetics 2002

3. 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

4. Probing fibroblast biology in the mammalian skin

5. Activated Fibroblasts in Tissue Repair and Disease
Wound healing
Adapted from:
Kalluri and Zeisberg 2006
Koontongkaew 2013
Tissue Fibrosis
Hyperactive
Myofibroblast
Tumor Stroma
CAF
Inflammation
Proliferation
Remodeling

6. Myofibroblast Differentiation in Skin Wounds & Cancer

7. Scar formation in wounded Snail transgenic skin
Human skin
Mouse skin

8. Snail is involved in development and disease

9. K14
Snail
(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

10. Expression profile of Snail in the skin
Jamora et al., 2004

11. Mikkola and Millar, 2006
Budding Morphogenesis Mimics Tumor Invasion

12. Expression profile of Snail in the skin
K14-Snail
Jamora et al., 2004

13. What is the function of Snail in hair bud
morphogenesis and cutaneous disease?Effect of Snail overexpression in the epidermis

14. Snail expression induces epidermal involution & hyperplasia
WT Snail Tg

15. Metastasis associated markers are expressed
in Snail transgenic epidermis

16. Snail induces cutaneous inflammation

17. Characteristics of activated fibroblasts
From Kalluri and Zeisberg 2006

18. Snail transgenic skin displays elevated dermal ECM
K5/fibronectinK5/collagen Snail TgWT

19. Equilibrium between ECM production
and turnover is disrupted

20. Snail transgenic mouse has thick skin
WT Snail Tg

21. K14
Snail
(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

22. How is epidermal Snail inducing dermal fibrosis?
epidermis
Snail
dermis

23. Active fibroblasts are not a product of an EMT
Guarino et al., Human Pathology 2009
β-gal/αSMA

24. 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 transgenic
keratinocytes

25. untreated WT skin CM
Snail Tg skin CM TGFβ2
%gelcontraction
untreated
WT
skin CM
Snail Tg
skin CM
TGFβ2
Secreted factor can activate dermal fibroblasts: gel contraction

26. Conditioned media from Snail epidermal explants
can induce expression of active fibroblast genes
0
5
10
15
20
25
mRNAexpression(relativefold)
WT Snail Tg Snail Tg +
IL1R KO df
Snail Tg +
TGFβ inhib
CTGF
αSMA
Conditioned media

27. TGFβ and tissue fibrosis

28. Clinical trials targeting TGFβ
Targeting the TGFβ signalling pathway in disease
Rosemary J. Akhurst & Akiko Hata
Nature Reviews Drug Discovery 11, 790-811 (October 2012)
Page 1 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html
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 Disease
applications
Stage Clinical trial
identifiers
Summary of
results
Refs
Trabedersen (AP12009);
Antisense Pharma
Antisense
oligo
TGFβ2
ligand
Glioblastoma Phase
I/IIb
NCT00431561 Safe 70,73,74
Pancreatic cancer,
MetM, colon
cancer
Phase I NCT00844064 Pancreatic cancer
trials continue
84
Glioblastoma Phase III NCT00761280 Glioblastoma trials
stopped in March
2012 owing to
advances in
standard of care
and neurosurgery
(BOX 4)
-
Belagenpumatucel-L
(Lucanix); NovaRx
Antisense
gene-
modified
allogeneic
tumour cell
vaccine
TGFβ2 NSCLC Phase III NCT00676507 Well tolerated in 75
patients; survival
advantage justifies
further Phase III
evaluation
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
Disitertide (P144); Digna
Biotech
Peptide Peptide
based on
TβRIII
that
blocks
ligand
binding
to
receptors
Skin fibrosis in
systemic sclerosis
Phase II NCT00574613,
NCT00781053
Preclinical efficacy
in peritoneal
fibrosis associated
with peritoneal
dialysis, renal and
cardiac fibrosis,
corneal haze and
retinal AMD; safety
and efficacy in
Phase IIa clinical
trial for
scleroderma/skin
fibrosis
75,76,77
Lerdelimumab (CAT-152);
Cambridge Antibody
Technology
Humanized
antibody
TGFβ2
ligand
Reduction of
scarring after
glaucoma surgery
Phase III
(complete)
- Safe; ineffective in
reducing scarring
in Phase III trial
88,89
Metelimumab (CAT-192);
Cambridge Antibody
Technology
Humanized
Antibody
TGFβ1
ligand
Diffuse systemic
sclerosis
Phase I/II NCT00043706 Ineffective when
systemically
administered in
doses up to 10 mg
per kg
90
Fresolimumab (GC-1008);
Cambridge Antibody
Technology/Genzyme/Sanofi
Humanized
antibody
TGFβ1,
TGFβ2
and
TGFβ3
ligands
Focal segmental
glomerulosclerosis
Phase I NCT00464321 Completed and
safe; plans to
progress
92
Systemic sclerosis Phase I NCT01284322 Still recruiting -
Study ongoing -
Completed, no
results
-

29. What is in secreted by Snail expressing
keratinocytes to activate dermal fibroblasts?
epidermis
dermis
dermis
Snail
?
Collagen contraction activity
Induction of CTGF & αSMA
Concanavalin A sepharose column
Collagen contraction activity
Induction of CTGF & αSMA
WT or Snail Tg Conditioned media
Anion exchange chromatography (Mono Q)
Mass
Spec

30. Candidate proteins mediating
epithelial-mesenchymal crosstalk

31. 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

32. 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

33. Mindin activates NFκB in dermal fibroblasts
dermal
fibroblasts
(NFκB)
Control + mindin
0
4
8
12
16
20
Control Mindin
Relativelevelof
RANTESRNA
WT
FN
pNFkB
K5
epi
epi
der
epi
der
epi
der
der
hf
A Snail Tg
WT Snail Tg

34. Spondin-2/mindin activates NFκB in dermal fibroblasts
0
5
10
15
20
25
Control Spondin-2
ActivityofNFκB-lucreporter
Intrinsic Gene Expression Subsets of Diffuse
Cutaneous Systemic Sclerosis Are Stable in Serial
Skin Biopsies
Sarah A. Pendergrass1
, Raphael Lemaire2
, Ian P. Francis2
, J. Matthew Mahoney1
, Robert Lafyatis2
and
Michael 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 9
healthy controls. These data recapitulate the patient ‘‘intrinsic’’ gene expression subsets described previously,
including fibroproliferative, inflammatory, and normal-like groups. Serial skin biopsies showed consistent and
non-progressing gene expression over time, and importantly, the patients in the inflammatory subset do not
move to the fibroproliferative subset, and vice versa. We were unable to detect significant differences in gene
expression before and after rituximab treatment, consistent with an apparent lack of clinical response. Serial
biopsies from each patient stayed within the same gene expression subset, regardless of treatment regimen or
ORIGINAL ARTICLE
Category Term
GOTERM_BP_ALL GO:0002376~immune system process
GOTERM_BP_ALL GO:0006955~immune response
GOTERM_BP_ALL GO:0050896~response to stimulus
Functional Group 2
Category Term
GOTERM_BP_ALL GO:0009611~response to wounding
GOTERM_BP_ALL GO:0006950~response to stress
GOTERM_BP_ALL GO:0009605~response to external stimulus
GOTERM_BP_ALL GO:0006952~defense response
GOTERM_BP_ALL GO:0006954~inflammatory response
Functional Group 3
Category Term
GOTERM_BP_ALL GO:0032502~developmental process
GOTERM_BP_ALL GO:0048513~organ development
GOTERM_BP_ALL GO:0007275~multicellular organismal development
GOTERM_BP_ALL GO:0048856~anatomical structure development
GOTERM_BP_ALL GO:0048731~system development
GOTERM_BP_ALL GO:0032501~multicellular organismal process
Category Term
GOTERM_BP_ALL GO:0006915~apoptosis
GOTERM_BP_ALL GO:0012501~programmed cell death
GOTERM_BP_ALL GO:0008219~cell death
GOTERM_BP_ALL GO:0016265~death
GOTERM_BP_ALL GO:0048468~cell development
GOTERM_BP_ALL GO:0042981~regulation of apoptosis
GOTERM_BP_ALL GO:0043067~regulation of programmed cell death
GOTERM_BP_ALL GO:0048869~cellular developmental process
GOTERM_BP_ALL GO:0030154~cell differentiation
Category Term
GOTERM_BP_ALL GO:0006928~cell motility
GOTERM_BP_ALL GO:0051674~localization of cell
GOTERM_BP_ALL GO:0016477~cell migration
Category Term
GOTERM_BP_ALL GO:0006817~phosphate transport
GOTERM_BP_ALL GO:0015698~inorganic anion transport
GOTERM_BP_ALL GO:0006820~anion transport
Category Term
GOTERM_BP_ALL GO:0007243~protein kinase cascade
GOTERM_BP_ALL GO:0009966~regulation of signal transduction
GOTERM_BP_ALL GO:0009967~positive regulation of signal transduction
GOTERM_BP_ALL GO:0007249~I-kappaB kinase/NF-kappaB cascade
GOTERM_BP_ALL GO:0043123~positive regulation of I-kappaB kinase/NF-kappaB cascade
GOTERM_BP_ALL GO:0043122~regulation of I-kappaB kinase/NF-kappaB cascade
From JID 2012
Supp. Fig. 3

35. Mindin contributes to inflammation
K5
Mac1
K5
Mac1
K5
Mac1
K5
Mac1
K14Snail – Mindin +/- K14Snail – Mindin -/-
K14Snail + Mindin +/- K14Snail + Mindin -/-
0
5
10
15
20
25
30
35
40
45
WT Snail Snail +
mindin KO
#macrophages/frame
Snail+ keratinocytes
mindin
dermal
fibroblasts
Secretion of
Proinflammatory
cytokines
X

36. Immunosuppression blocks epidermal
hyperplasia and involution
WT Snail Tg
Snail Tg +
anti-inflammatory

37. K14Snail- Mindin +/- K14Snail- Mindin -/-
K14Snail+ Mindin +/- K14Snail+ Mindin -/-
Mindin contributes to epidermal hyperplasia
0
10
20
30
40
50
60
70
WT Snail Snail +
mindin KO
epidermalthickness(mm)

38. Does mindin contributes to dermal thickening?
Wynn & Ramalingam 2012

39. Mindin contributes to dermal thickening
0
10
20
30
40
50
60
70
WT Snail Snail + mindin
KO
dermalthickness(mm)

40. Snail promotes dermal fibroblast crosstalk
mindin
adapted from Kalluri and Zeisberg 2006

41. 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)

42. Clinical trials targeting TGFβ
Targeting the TGFβ signalling pathway in disease
Rosemary J. Akhurst & Akiko Hata
Nature Reviews Drug Discovery 11, 790-811 (October 2012)
Page 1 of 6http://www.nature.com/nrd/journal/v11/n10/fig_tab/nrd3810_T1.html
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 Disease
applications
Stage Clinical trial
identifiers
Summary of
results
Refs
Trabedersen (AP12009);
Antisense Pharma
Antisense
oligo
TGFβ2
ligand
Glioblastoma Phase
I/IIb
NCT00431561 Safe 70,73,74
Pancreatic cancer,
MetM, colon
cancer
Phase I NCT00844064 Pancreatic cancer
trials continue
84
Glioblastoma Phase III NCT00761280 Glioblastoma trials
stopped in March
2012 owing to
advances in
standard of care
and neurosurgery
(BOX 4)
-
Belagenpumatucel-L
(Lucanix); NovaRx
Antisense
gene-
modified
allogeneic
tumour cell
vaccine
TGFβ2 NSCLC Phase III NCT00676507 Well tolerated in 75
patients; survival
advantage justifies
further Phase III
evaluation
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
Disitertide (P144); Digna
Biotech
Peptide Peptide
based on
TβRIII
that
blocks
ligand
binding
to
receptors
Skin fibrosis in
systemic sclerosis
Phase II NCT00574613,
NCT00781053
Preclinical efficacy
in peritoneal
fibrosis associated
with peritoneal
dialysis, renal and
cardiac fibrosis,
corneal haze and
retinal AMD; safety
and efficacy in
Phase IIa clinical
trial for
scleroderma/skin
fibrosis
75,76,77
Lerdelimumab (CAT-152);
Cambridge Antibody
Technology
Humanized
antibody
TGFβ2
ligand
Reduction of
scarring after
glaucoma surgery
Phase III
(complete)
- Safe; ineffective in
reducing scarring
in Phase III trial
88,89
Metelimumab (CAT-192);
Cambridge Antibody
Technology
Humanized
Antibody
TGFβ1
ligand
Diffuse systemic
sclerosis
Phase I/II NCT00043706 Ineffective when
systemically
administered in
doses up to 10 mg
per kg
90
Fresolimumab (GC-1008);
Cambridge Antibody
Technology/Genzyme/Sanofi
Humanized
antibody
TGFβ1,
TGFβ2
and
TGFβ3
ligands
Focal segmental
glomerulosclerosis
Phase I NCT00464321 Completed and
safe; plans to
progress
92
Systemic sclerosis Phase I NCT01284322 Still recruiting -
Study ongoing -
Completed, no
results
-

43. TGFβ and tissue fibrosis
Failure in clinical trials

44. untreated WT skin CM
Snail Tg skin CM TGFβ2
%gelcontraction
untreated
WT
skin CM
Snail Tg
skin CM
TGFβ2
Secreted factor can activate dermal fibroblasts: gel contraction

45. Snail is sufficient to activate dermal fibroblasts
Tubulin
Snail
untransfected
K14vector
K14-Snail
Transfection of
Primary keratinocytes
Effect of CM on
Dermal fibroblasts (contraction)
0
10
20
30
40
50
Untransfected K14 vector K14-Snail
%gelcontraction
CM from transfected keratinocytes
Untreated CM
95oC CM

46. Cancer associated fibroblasts
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, cellAdapted from Cirri and Chiarugi, Am J. Cancer Res 2011
SNAIL
Mindin
cell proliferation
inflammation
Snail expressing keratinocytes activate CAFs

47. 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
Cell. 2012 Mar 2;148(5):1015-28.
Slug and Sox9 cooperatively determine the mammary stem cell
state.
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.
Display Settings: Abstract
PubMed
Du et al., 2010