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Bone Metastasis in Breast Cancer:
Molecular Pathogenesis
Maurizio Longo
Dip. Medicina Sperimentale, Università dell’Aquila
Highlights in the Management of Breast Cancer
Roma, Domus Sessoriana, Nov. 16-17 2006
Bone Metastasis: a Most Severe Complication
of Breast Cancer
• Up to 80% of patients with metastatic breast cancer
will develop bone metastases
• Long-term survivor patients with only
metastasis undergo very poor life quality
bone
Bone-Metastatic Disease of Breast Cancer:
Important Symptoms
• Hyper-calcemia
• Pain, often disabling and resistant to palliation
• Pathologic fracture
• (Breast carcinomas account for >50% of the met.
cases requiring orthopedic intervention)
Bone-Metastatic Disease of Breast Cancer:
Clinical Features
• Multifocal: at autopsy, nearly all patients demonstrate
multiple, small “asymptomatic” bone metastases
• Preferably located to the axial skeleton, together with active
hemopoietic marrow
• Indolent course (e.g. remarkable progression-free periods
after treatment)
Osteolysis and Pathologic Fracture
Lytic areas
Mechanical properties are severely endangered also by “mixed”
lesion (lytic/blastic)
METASTASIS: a Multi-step Process (I)
• Detachment from tumour
(dependent on E-M transition)
• Transport in the bloodstream
(estimated <0.1% surviving this phase)
• Adhesion to endothelial cells
(mostly random*)
• Invasion of host tissue
*Debated
Target-independ.
METASTASIS: a Multi-step Process (II)
Target-dependent steps:
1. “Colonisation” of tissue
2. Triggering of growth
(cross-talk with the host tissue)
3. Independent growth of secondary tumour
(only autocrine and systemic regulation)
The Metastasis Process: “Classical” Description
An “obsolete” model?
Main points presently challenged of the
“classical” description:
1. Cell detachment as a late event
2. “Unbridled” growth of (micro)metastases
--> In fact, recent evidence obtained in in-vivo models shows:
1) single tumour cells in distant tissues during earliest
development phases of metastatic tumours;
2) extremely slow development of many micrometastases.
Growth of micrometastases is a
target-dependent process
The Bone Micro-environment:
Main Cell Players
The bone organ is also comprised of: marrow stromal cells,
hemopoietic cells, adipocytes. (Fibrous tissue may also appear.)
Osteoblasts and Osteoclasts: Main Features
• Osteoblasts (OBs) develop from mesenchimal cells
• Osteoclasts (OCs) develop from the fusion of blood cells of the
immune system, the monocytes
• OBs are bone-residing cells
• OC precursors are recruited from the bloodstream
• OBs proliferate, OCs do not. The OC life-span is limited
(apoptosis can only be delayed)
Both OBs and Ocs are polarised, very actively secreting cells:
• OBs secrete bone matrix glycoproteins and collagen
• OCs secrete HCl and lytic enzymes, and digest collagen
The Bone Remodeling Unit (I)
“Reversal” cells
OBs
OCs
Resorption cone. (Early osteoblasts do not adhere to the bone
surface and proliferate actively.)
The Bone Remodeling Unit (II)
Resorption is fast, apposition is slow, mineralisation slower.
I.e. --> uncontrolled resorption can damage bone fast!
The resorption activity of osteoclasts is
potentially very harmful and is finely regulated.
What controls osteoclasts?
Bone Resorption by Osteoclasts:
Multiple Control Levels
• Systemic: hormones (incl. Vitamin D3) and metabolic
signals
• Local:
• Secreted factors (chemokines,
cytokines)
• Cell-cell interactions
interleukins,
Note well: at the local level, phisiological
regulation relies crucially on:
osteoblasts and their precursors.
Osteoclast control: Local Factors
SDF-1
M-CSF
RANKL
OPG
Vitamin D3
PTH/PTHrP
IL-6
Chemotaxis
Proliferation
Fusion
Differentiation
Resorption
Apoptosis
Vitamin D3
IL-1, IL-6
Osteoclast control: Cell-cell Interaction
OPG
RANKL
RANK
M-CSF
c-Fms
Key point:
Bone met. cells are, or become*, highly
responsive to many signals destined to bone cells.
*Or both!
What survival and growth signals for bone cells
do met. cells exploit ?
1. Survival: Stem Cell Niches
Micrometastasis cell originate from (actively) dividing tumour
cells with unlimited division potential
Despite this,
development
micrometastasis
show
an
indolent
initial
ANALOGY:
Stem cells are characterised by unlimited division potential
BUT
stem cells divide with extremely low frequency
The “Cancer Stem Cell” Hypothesis:
Tumours originate from stem/progenitor cells of the host tissue
Normal tissues
Carcinomas, etc.
Adenomas,
sarcomas, etc.
The Hemopietic Stem Cell Niche in Bone (I)
Niche-dependent differentiation in healthy bone
Stem Cell Niche in Bone:
Overview of Present Knowledge
• Formed by SNO cells, an osteoblastic sub-population
• SNO cells are:
• Spindle-shaped (poorly polarised / non-secreting)
• N-Cadherin-positive
• CD45-negative
Stem Cell Niche in Bone:
Overview of Present Knowledge
• Formed by SNO cells, an osteoblastic sub-population
• SNO cells are:
• Spindle-shaped (poorly polarised / non-secreting)
• N-Cadherin-positive
• CD45-negative
• SNO cells maintain self-renewing long-term stem cells in a
semi-quiescent state
• Thus, activation of stem cells requires detachment from SNO
cells
Stem Cell Niche in Bone:
Overview of Present Knowledge
• Formed by SNO cells, an osteoblastic sub-population
• SNO cells are:
• Spindle-shaped (poorly polarised / non-secreting)
• N-Cadherin-positive
• CD45-negative
• SNO cells maintain self-renewing long-term stem cells in a
semi-quiescent state
• Activation of stem cells requires detachment from SNO cells
• Detachment from the niche is favoured by various phisiological
(e.g. chemotactic) and pathological signals
The Cancer Stem Cell Niche in Bone
Hypothesis:
Once in the bone marrow micro-environment,
cancer stem cell may achieve long survival by
interacting with stem-cell niches
The Cancer Stem Cell Niche in Bone
Hypothesis:
Once in the bone marrow micro-environment,
cancer stem cell may achieve long survival by
interacting with stem-cell niches
On the other hand, the niche will prevent cancer stem cells from
proliferate appreciably! <-- Explanation of metastasis indolence
Additional signals can lead to further metastasis progression
The Hemopietic Stem Cell Niche in Bone (II)
Abnormal niche-dep. differentiation in metastasis-bearing bone
2. Growth:
The Initial Development of Metastases
or:
«Spies in Enemy Headquarters»
How can an ex-epithelial cell manage to live
well once free in the bone marrow?
• Initial metastases (few cancer cells) cannot rely on autocrine
growth-promoting (and anti-apoptosis) signals
• Thus, only metastasis cells responding best to available growth
signals will undergo positive selection
• Of these, cells also capable of over-stimulating local bone
resorption will be further favoured (due to creation of larger
space for growth)
The Metastasis “Vicious Circle” (I)
The Metastasis “Vicious Circle” (II)
Bone Matrix
OBs
RankL-Rank
PTHrP, etc.
Pre-OCs
Bone
Resorption
OCs
Proliferation
Survival
Migration
TGF-
FGFs
PDGF
IGF-1
BMPs
IL-6, TNFα,
M-CSF, PGE2
Cancer cells
Observation:
Beside releasing same OC-genic factors as OBs,
various bone met. cells also express bone-cell
markers (e.g. BSP, OPN) and transcription factors
(e.g. Runx2).
Observation:
Beside releasing same OC-genic factors as OBs,
various bone met. cells also express bone-cell
markers (e.g. BSP, OPN) and transcription factors
(e.g. Runx2).
Just a coincidence?
How can an epithelial cell manage to live well in
the bone marrow?
• Initial metastases (few cancer cells) cannot rely on autocrine
growth-promoting (and anti-apoptosis) signals
• Thus, only metastasis cells responding best to available growth
signals will undergo positive selection
• Of these, cells also capable of over-stimulating local bone
resorption will be further favoured (due to creation of larger
space for growth)
Result: various metastasis cells will eventually
“resemble”* the bone-residing cells governing
bone resorption (i.e. OBs!)
OR
The Met. “Osteomimicry” Hypothesis
*Pre-adaptation also possible
Met. Osteomimicry: Some Evidence
•
Lin DL, Tarnowski CP, Zhang J, Dai J, Rohn E, Patel AH, Morris MD, Keller ET.
Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in
vitro.Prostate. 2001 May 15;47(3):212-21.
•
Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, Bellahcene A, Van Wijnen
AJ, Young MF, Lian JB, Stein GS, Gerstenfeld LC.
Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the
expression of bone sialoprotein in human metastatic breast cancer cells. Cancer Res. 2003
May 15;63(10):2631-7.
•
Zhang JH, Tang J, Wang J, Ma W, Zheng W, Yoneda T, Chen J.
Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells
in a mouse model. Int J Oncol. 2003 Oct;23(4):1043-8.
•
Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB, Stein GS, Gerstenfeld LC.
Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastasesassociated osteolytic disease. Cancer Res. 2004 Jul 1;64(13):4506-13.
Transcriptome Analysis:
Genes more expressed in bone mets versus nonbone-mets: any bone-cell markers?
YES!
Transcriptome Analysis:
The Osteomimicry Signature
Gene symbol
Ratio bone mets
vs non-bone mets
Bone mets
vs primary
Ratio bone mets
vs normal bone
MMP-9
32
12
2.21
ITGB3
2.0
4.4
0.63
CTSK
16
1.5
0.82
IBSP
11
31
0.25
OMD
37
3.4
0.36
RAMP-2
2.7
1.8
0.23
MEOX-2
4.7
2.8
0.37
IGF-BP5
4.3
4.4
0.91
The Osteomimicry Signature:
OB Genes*
Gene symbol
Ratio bone mets
vs non-bone mets
Bone mets
vs primary
Ratio bone mets
vs normal bone
MMP-9
32
12
2.21
ITGB3
2.0
4.4
0.63
CTSK
16
1.5
0.82
IBSP
11
31
0.25
OMD
37
3.4
0.36
RAMP-2
2.7
1.8
0.23
MEOX-2
4.7
2.8
0.37
IGF-BP5
4.3
4.4
0.91
*Shown only cSrc-regulated genes
The Osteomimicry Signature:
OC Genes
Gene symbol
Ratio bone mets
vs non-bone mets
Bone mets
vs primary
Ratio bone mets
vs normal bone
MMP-9
32
12
2.21
ITGB3
2.0
4.4
0.63
CTSK
16
1.5
0.82
IBSP
11
31
0.25
OMD
37
3.4
0.36
RAMP-2
2.7
1.8
0.23
MEOX-2
4.7
2.8
0.37
IGF-BP5
4.3
4.4
0.91
Hypothesis (I):
The osteomimicry “strategies”* of bone
metastasis cells relies not only on osteoblast-, but
also on osteoclast-like features
*Multiple osteomimetic pseudo-phenotypes possible
Hypothesis (II):
Part of the observed pseudo-osteoclastic
phenotype of bone met. cells might accomplish
“micro-resorption”.
Hypothesis (II):
Part of the observed pseudo-osteoclastic
phenotype of bone met. cells might accomplish
“micro-resorption”.
Micro-resorption could be the trigger of the
metastasis vicious circle.
Thank You for your attention!