Presentazione di PowerPoint

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Bone Metastasis in Breast Cancer Molecular
Pathogenesis
Maurizio Longo Dip. Medicina Sperimentale,
Università dellAquila
Highlights in the Management of Breast
Cancer Roma, Domus Sessoriana, Nov. 16-17 2006
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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 bone
  metastasis undergo very poor life quality

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Bone-Metastatic Disease of Breast
Cancer Important Symptoms

• Hyper-calcemia
• Pain, often disabling and resistant to
  palliation
• Pathologic fracture
• (Breast carcinomas account for gt50 of the met.
  cases requiring orthopedic intervention)

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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)

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Osteolysis and Pathologic Fracture
Lytic areas
Mechanical properties are severely endangered
also by mixed lesion (lytic/blastic)
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METASTASIS a Multi-step Process (I)
Debated
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METASTASIS a Multi-step Process (II)

• Target-dependent steps
• Colonisation of tissue
• Triggering of growth
• (cross-talk with the host tissue)
• Independent growth of secondary tumour
• (only autocrine and systemic regulation)

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The Metastasis Process Classical Description
An obsolete model?
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Main points presently challenged of the
classical description

• Cell detachment as a late event
• Unbridled growth of (micro)metastases
• --gt 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.

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Growth of micrometastases is a target-dependent
process
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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.)
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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

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The Bone Remodeling Unit (I)
Resorption cone. (Early osteoblasts do not adhere
to the bone surface and proliferate actively.)
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The Bone Remodeling Unit (II)
Resorption is fast, apposition is slow,
mineralisation slower. I.e. --gt uncontrolled
resorption can damage bone fast!
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The resorption activity of osteoclasts is
potentially very harmful and is finely
regulated. What controls osteoclasts?
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Bone Resorption by Osteoclasts Multiple Control
Levels

• Systemic hormones (incl. Vitamin D3) and
  metabolic signals
• Local
• Secreted factors (chemokines, interleukins,
  cytokines)
• Cell-cell interactions

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Note well at the local level, phisiological
regulation relies crucially on osteoblasts and
their precursors.
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Osteoclast control Local Factors
SDF-1 M-CSF RANKL OPG
Chemotaxis Proliferation Fusion Differentiation Re
sorption Apoptosis
Vitamin D3 IL-1, IL-6
Vitamin D3 PTH/PTHrP IL-6
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Osteoclast control Cell-cell Interaction
OPG
RANK
RANKL
c-Fms
M-CSF
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Key point Bone met. cells are, or become,
highly responsive to many signals destined to
bone cells.
Or both!
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What survival and growth signals for bone cells
do met. cells exploit ?
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1. Survival Stem Cell Niches
Micrometastasis cell originate from (actively)
dividing tumour cells with unlimited division
potential Despite this, micrometastasis show an
indolent initial development
ANALOGY
Stem cells are characterised by unlimited
division potential BUT stem cells divide with
extremely low frequency
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The Cancer Stem Cell Hypothesis Tumours
originate from stem/progenitor cells of the host
tissue
Adenomas, sarcomas, etc.
Normal tissues
Carcinomas, etc.
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The Hemopietic Stem Cell Niche in Bone (I)
Niche-dependent differentiation in healthy bone
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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

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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

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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

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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
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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! lt--
Explanation of metastasis indolence Additional
signals can lead to further metastasis progression
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The Hemopietic Stem Cell Niche in Bone (II)
Abnormal niche-dep. differentiation in
metastasis-bearing bone
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2. Growth
The Initial Development of Metastases or Spies
in Enemy Headquarters
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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)

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The Metastasis Vicious Circle (I)
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The Metastasis Vicious Circle (II)
Bone Matrix
RankL-Rank
IL-6, TNFa, M-CSF, PGE2
PTHrP, etc.
OBs
Pre-OCs
Bone Resorption
OCs
Proliferation Survival Migration
TGF-?
FGFs
PDGF
IGF-1
Cancer cells
BMPs
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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).
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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?
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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)

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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
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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 1547(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
  1563(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 Oct23(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
  metastases-associated osteolytic disease. Cancer
  Res. 2004 Jul 164(13)4506-13.

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Transcriptome Analysis Genes more expressed in
bone mets versus non-bone-mets any bone-cell
markers? YES!
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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
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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
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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
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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
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Hypothesis (II)Part of the observed
pseudo-osteoclastic phenotype of bone met. cells
might accomplish micro-resorption.
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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.
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Thank You for your attention!