StromalEpithelial Interactions in Cancer

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Stromal-Epithelial Interactions in Cancer
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(SV)
SVM
UGM
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Paracrine Signaling

• A cell or tissue produces a factor which acts
  upon an adjacent tissue.
• Examples include many growth factors during
  development, adult homeostasis and in cancer.

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Sub-renal Capsule Grafting
Brody, Young and Cunha http//mammary.nih.gov/tool
s/mousework/Cunha001/index.html
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Androgens
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Stromal-epithelial interactions play a key role
in prostatic development.

• Mesenchyme
• Specifies prostatic epithelial identity
• Induces prostatic bud formation
• Elicits bud elongation
• Induces and specifies patterns of ductal
  branching
• Regulates prostatic epithelial growth
• Regulates epithelial apoptosis
• Specifies lobar functional identity

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Androgens acting on developing prostate
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Androgens acting on normal adult prostate
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Stromal biology in a glandular organ
Secretory Cells
Epithelium
Basal Cells
Basement Membrane

• Fibroblasts

ECM

• SMC

Stroma
Growth Factors (latent)
Blood Vessels

• Structural and Regulatory

Immune Cells
Nerves
Wound repair to maintain tissue homeostasis
David Rowley, BCM
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Stromal changes in carcinogenesis
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Androgen/stromal/epithelial axis in a nascent
prostate tumor
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Androgen/stromal/epithelial axis in locally
growing prostate cancer
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Stromal Response is Induced During PIN
vimentin
vimentin/a-actin
pro-collagen I
trichrome
David Rowley, BCM
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Massons Trichrome Stain of Human Prostate
David Rowley, BCM
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Stromal Cell Phenotype in Normal Prostate
vimentin
calponin
a-actin
David Rowley, BCM
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Stromal Cell Phenotype in Prostate Cancer
vimentin
calponin
a-actin
David Rowley, BCM
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Stromal Response in Wound Repair
------ Reactive Stroma

• Wound Repair Response
• Stromal cell activation to myofibroblast /
  fibroblast phenotype
• ECM production - remodeling / Remodeling
  enzymes
• Expression of growth factors / Altered
  bioavailability
• Angiogenesis
• Re-epithelialization

FGF family TGF-b family PDGF family
David Rowley, BCM
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Isolation of Prostatic Fibroblasts
Collagenase/ Hyaluronidase
CAF
NPF
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CAFBPH-1
NPFBPH-1
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Signaling Mechanisms involved in paracrine
interactions promoting cancer a short tale of
two intersecting pathways
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TGF-b in normal prostate and in prostate cancer
tissues

• Normal
• Maintenance of smooth muscle differentiation.
• Restriction of epithelial proliferation.
• Cancer
• Bioavailable TGF-b levels increased.
• Local Immunosuppression.
• Reactive Stroma.
• Increased Angiogenesis.
• Epithelial to mesenchymal transformation.
• Increased epithelial cell motility and invasion.

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TGF-ß
Smad 7
P
Smad 2
RhoA
P
Smad 3
Smad 4
ROCK
TAK1
P
PKN
Smad 2/3
myc
Smad 4
MKK3/6
p27
cyE
Cdk2
p15
p38
Rb
cyD
Cdk4/6
E2F
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Consequences on response to CAF stimulation of
reducing epithelial TGF-beta response.
BPH1-DNCAF
BPH1-EVCAF
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Consequence of blocking TGF? ligand on response
to CAF
2G7 Treatment
Control
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Smad2 Phosphorylation
2G7 Treatment
Control
HE
P-Smad2
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• TGF-beta inhibits the proliferation of BPH1
  cells.
• BUT
• TGF-beta is a critical component in tumorigenesis
  elicited by CAF.
• It is counterintuitive that deletion of a growth
  inhibitory pathway would directly suppress
  tumorigenesis.
• How can we resolve this?

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CXCL12/SDF1 Expression by CAF and NPF
350.0
300.0
250.0
200.0
SDF1(ng/ml)
150.0
100.0
50.0
0.0
CAF
NPF
Conditioned Medium
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CXCL12/SDF1 signals through CXCR4, a G-protein
coupled receptor which activates a number of
pathways including Ras/Erk and PI3K/Akt.
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CXCR4 In Tissue Recombinants
BPH1UGM
BPH1CAF
H E
CXCR4
PI

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CXCR4 expression in BPH1 cells following exposure
to TGF-?
24h
48h
72h
Control
72hr TGFß
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CXCR4 Staining in BPH1CAF Tissue Recombinant
when TGF-? Signaling is Blocked
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Consequences on response to CAF stimulation of
reducing epithelial CXCR4.
shRNA control
CXCR4shRNA-1
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• TGF-beta is a critical component in tumorigenesis
  elicited by CAF.
• TGF-beta, CAF cells and CAF conditioned medium
  can all promote expression of CXCR4 in initiated
  BPH1 cells.
• SDF1/CXCR4 signaling is required for
  tumorigenesis.

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In initiated but non-tumorigenic cells (BPH1)
TGF-beta and CXCL12 cooperate to elicit changes
compatible with tumor progression in vivo.
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Are things likely to be this simple?
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Activated c-Met
BPH1 CAF 2G7 Treated
BPH1 CAF
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Is it possible to modify stromal cells so that
they behave like tumor stroma?
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Conditional Knockout of the Type II TGF-ß
Receptor
Tgfbr2floxE2
LoxP
LoxP
Cre
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LoxP
LoxP
Cre
Science 2004, 303848
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Prostate
Tgfbr2fspko
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Proliferation in the Absence of Androgens
Following 4 days castration
Ki67
Tgfbr2flox/flox
Tgfbr2flox/flox
Tgfbr2fspko
Tgfbr2fspko
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Interpretation?
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Loss of stromal TGFßR function leads to
incomplete stromal differentiation and failure to
modify paracrine interactions to a normal growth
quiescent adult situation.
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• The FSP promoter - which drives cre recombinase
  expression is not expressed in every stromal
  cell. In fact only 30-40 of stromal cells in
  these mice knock out TGFßRII

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Juxtacrine Signaling

• A cell produces something which acts upon cells
  in direct contact with or extremely close to the
  source.
• Could be a soluble factor which travels outside
  of the cells or something which is transferred
  through gap junctions.
• Examples include drug resistance proteins.

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TGFb signaling in prostate stromal cells regulate
tumor suppressive and tumor promoting factors
Flox 100 FbKO 0
Flox 50 FbKO 50
Flox 0 FbKO 100
Neil Bhowmick
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Interpretation?
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Things to consider regarding the microenvironment

• Complexity multiple cell types, such as
• Epithelium
• Muscle/fat
• Fibroblasts
• Bone marrow derived cells
• Signaling mechanisms include autocrine,
  juxtacrine, paracrine, exocrine and endocrine -
  including combinations of these.
• Some signals are unidirectional but most are not.
• Even these classically paracrine pathways can be
  subverted to an autocrine function in cancer.
• Many signaling pathways interact.

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Bierie et al. Nature Reviews Cancer 6, 506520
(July 2006) doi10.1038/nrc1926
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Graft Histology
BPH1DN CAF
BPH1 CAF
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Graft Histology
BPH1GFPshRNA CAF
BPH1CXCR4shRNA CAF